Antagonists of the bradykinin B1 receptor

ABSTRACT

The present invention relates to certain biologically active peptides and conjugated peptides which can be used as therapeutics or prophylactics against diseases or conditions linked to B1 as the causative agent. In a preferred embodiment of the invention biologically active PEG-conjugated peptides are provided. In one aspect of the present invention, pharmacologically active PEG-conjugated peptides of the present invention are useful to treat inflammation or pain.

This application claims the benefit of U.S. Provisional Application No.60/513,913 filed Oct. 22, 2003, and U.S. Provisional Application No.60/538,929 filed Jan. 24, 2004, which are hereby incorporated byreference.

BACKGROUND OF THE INVENTION

More than two million people in the United States alone areincapacitated by chronic pain on any given day (T. M. Jessell & D. D.Kelly, Pain and Analgesia in PRINCIPLES OF NEURAL SCIENCE, third edition(E. R. Kandel, J. H. Schwartz, T. M. Jessell, ed., (1991)).Unfortunately, current treatments for pain are only partially effective,and many also cause life-style altering, debilitating, and/or dangerousside effects. For example, non-steroidal anti-inflammatory drugs(“NSAIDs”) such as aspirin, ibuprofen, and indomethacin are moderatelyeffective against inflammatory pain but they are also renally toxic, andhigh doses tend to cause gastrointestinal irritation, ulceration,bleeding, increased cardiovascular risk, and confusion. Patients treatedwith opioids frequently experience confusion and constipation, andlong-term opioid use is associated with tolerance and dependence. Localanesthetics such as lidocaine and mixelitine simultaneously inhibit painand cause loss of normal sensation. In addition, when used systemicallylocal anesthetics are associated with adverse cardiovascular effects.Thus, there is currently an unmet need in the treatment of chronic pain.

Pain is a perception based on signals received from the environment andtransmitted and interpreted by the nervous system (for review, seeMillan, M. J., The induction of pain: an integrative review. ProgNeurobiol 57:1-164 (1999)). Noxious stimuli such as heat and touch causespecialized sensory receptors in the skin to send signals to the centralnervous system (“CNS”). This process is called nociception, and theperipheral sensory neurons that mediate it are nociceptors. Depending onthe strength of the signal from the nociceptor(s) and the abstractionand elaboration of that signal by the CNS, a person may or may notexperience a noxious stimulus as painful. When one's perception of painis properly calibrated to the intensity of the stimulus, pain serves itsintended protective function. However, certain types of tissue damagecause a phenomenon, known as hyperalgesia or pronociception, in whichrelatively innocuous stimuli are perceived as intensely painful becausethe person's pain thresholds have been lowered. Both inflammation andnerve damage can induce hyperalgesia. Thus, persons afflicted withinflammatory conditions, such as sunburn, osteoarthritis, colitis,carditis, dermatitis, myositis, neuritis, inflammatory bowel disease,collagen vascular diseases (which include rheumatoid arthritis andlupus) and the like, often experience enhanced sensations of pain.Similarly, trauma, surgery, amputation, abscess, causalgia, collagenvascular diseases, demyelinating diseases, trigeminal neuralgia, cancer,chronic alcoholism, stroke, thalamic pain syndrome, diabetes, herpesinfections, acquired immune deficiency syndrome (“AIDS”), toxins andchemotherapy cause nerve injuries that result in excessive pain.

As the mechanisms by which nociceptors transduce external signals undernormal and hyperalgesic conditions become better understood, processesimplicated in hyperalgesia can be targeted to inhibit the lowering ofthe pain threshold and thereby lessen the amount of pain experienced.

Bradykinin (BK) and the related peptide, kallidin (Lys-BK)(see Table 3)mediate the physiological actions of kinins on the cardiovascular andrenal systems. However, the active peptides, BK and kallidin, arequickly degraded by peptidases in the plasma and other biological fluidsand by those released from a variety of cells, so that the half-life ofBK in plasma is reported to be approximately 17 seconds (1). BK andkallidin are rapidly metabolized in the body by carboxypeptidase N,which removes the carboxyterminal arginine residue to generate des-ArgBK or des-Arg kallidin. Des-Arg-kallidin is among the predominant kininsin man and mediate the pathphysiological actions of kinins in man. Inaddition to being a very potent proinflammatory peptide, des-Arg-BK ordes-Arg-kallidin is known to induce vasodilation, vascular permeability,and bronchoconstriction (for review, see Regoli and Barabe, Pharmacologyof Bradykinin and Related Kinins, Pharmacological Reviews, 32(1):1-46(1980)). In addition, des-Arg-BK and des-Arg-kallidin appear to beparticularly important mediators of inflammation and inflammatory painas well as being involved in the maintenance thereof. There is also aconsiderable body of evidence implicating the overproduction ofdes-Arg-kallidin in conditions in which pain is a prominent feature suchas septic shock, arthritis, angina, and migraine.

The membrane receptors that mediate the pleiotropic actions of kininsare of two distinct classes, designated B1 and B2. Both classes ofreceptors have been cloned and sequenced from a variety of species,including man (Menke, et al, Expression cloning of a human b1 bradykininreceptor. J. Biol. Chem. 269:21583-21586 (1994); Hess et al, Cloning andpharmacological characterization of a human bradykinin (BK-2) receptor.Biochem. Biophys. Res. Commun. 184, 260-268 (1992)). They are typical Gprotein coupled receptors having seven putative membrane spanningregions. In various tissues, BK receptors are coupled to every knownsecond messenger. B2 receptors, which have a higher affinity for BK,appear to be the most prevalent form of bradykinin receptor. Essentiallyall normal physiological responses and many pathophysio-logicalresponses to bradykinin are mediated by B2 receptors.

B1 receptors, on the other hand, have a higher affinity for des-Arg-BK(see Table 3) compared with BK, whereas des-Arg-BK is inactive at B2receptors. In addition, B1 receptors are not normally expressed in mosttissues. Their expression is induced upon injury or tissue damage aswell as in certain kinds of chronic inflammation or systemic insult(Marceau, F., et al., Kinin B1 receptors: a review. Immunpharmacology,30:1-26 (1995)). Furthermore, responses mediated by B1 receptors areup-regulated from a null level following administration of bacteriallipopolysaccharide (LPS) or inflammatory cytokines in rabbits, rats, andpigs (Marceau et al., (1998)).

The pain-inducing properties of kinins coupled with the inducibleexpression of B1 receptors make the B1 receptor an interesting target inthe development of anti-inflammatory, antinociceptive, antihyperalgesicand analgesic agents that may be directed specifically at injuredtissues with minimal actions in normal tissues. While a variety ofpeptide antagonists targeting the B1 receptor have been identified,their development as therapeutic analgesics has been stymied by poorefficacious half-lives resulting from very rapid degradation by tissueand serum peptidases and efficient renal clearance. More recently,peptide analogs having non-natural amino acid substituents have beenshown to be resistant to peptidases in in vitro stability assays (forreview, see Regoli et al, Bradykinin receptors and their antagonists.European Journal of Pharmacology, 348:1-10 (1998); Stewart, J. M., etal, Bradykinin antagonists: present progress and future prospects.Immunopharmacology, 43:155-161 (1999); and Stewart, J. M., et al.,Metabolism-Resistant Bradykinin Antagonists: Development andApplications. Biol. Chem., 382:37-41 (2001)).

Covalent conjugation of proteins with poly(ethylene glycol) (PEG) hasbeen widely recognized as an approach to significantly extend the invivo circulating half-lives of therapeutic proteins. PEGylation achievesthis effect predominately by retarding renal clearance, since the PEGmoiety adds considerable hydrodynamic radius to the protein (Zalipsky,S., et al., Use of functionalized poly(ethylene glycol)s formodification of polypeptides., in Poly(ethylene glycol) chemistry:Biotechnical and biomedical applications., J. M. Harris, Editor. (1992),Plenum Press: New York. p. 347-370.). Additional benefits oftenconferred by PEGylation of proteins include increased solubility,resistance to proteolytic degradation, and reduced immunogenicity of thetherapeutic polypeptide. The merits of protein PEGylation are evidencedby the commercialization of several PEGylated proteins includingPEG-Adenosine deaminase (Adagen™/Enzon Corp.), PEG-L-asparaginase(Oncaspar™/Enzon Corp.), PEG-Interferon α-2b(PEG-Intron™/Schering/Enzon), PEG-Interferon α-2a (PEGASYS™/Roche) andPEG-G-CSF (Neulasta™/Amgen) as well as many others in clinical trials.PEGylation of small therapeutic peptides, on the other hand, presentsunique challenges and has not been broadly applied. One of the greatestobstacles to peptide PEGylation is the essential requirement thatbiological activity be preserved in the final conjugate. Becausetherapeutic peptides often comprise the minimal sequence required foractivity and are therefore very small, they are relatively intolerant tosubstitution. PEG moieties are disproportionately larger than thepeptide itself and consequently are more likely to interfere stericallywith specific peptide:receptor binding interactions required foractivity. Thus, a peptide's ability to tolerate PEGylation and stillretain sufficient specific activity to be a useful therapeutic is quiteunpredictable and must be empirically determined (Morpurogo, et al.,Selective Alkylation and Acylation of α and ε Amino Groups with PEG in aSomatostatin Analogue: Tailored Chemistry for Optimized Bioconjugates.Bioconjugate Chem. 13:1238-1243 (2002)).

Clearly, there is a need for new, safe and effective treatments forinflammation and pain. It would be an advantage to have a B1 specificpeptide antagonist that is better able to tolerate systemic exposureduring treatment, by enhancing the circulating life (delayed clearance),solubility, stability, and/or decreasing the immunogenicity of themolecule. Increased circulating life would result in a less frequentdosing regimen and a less frequent dosing schedule would be moreconvenient to both physicians and patients, and would be particularlyhelpful to those patients involved in self-administration. Otheradvantages to less frequent dosing may include less drug beingintroduced into patients and increased compliance.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide novelbinding agents of the B1 receptor with demonstrably superiorpharmacokinetic properties in vivo as compared to known peptide B1antagonists yet sufficiently antagonize B1 receptor activity such thatthey are therapeutically useful in the treatment or prevention ofinflammation, pain, and other B1 mediated conditions including, but notlimited to, asthma and allergic rhinitis. Such agents are provided bythe present invention in the form of novel peptide antagonists andconjugated peptide antagonists of the B1 receptor. In one embodiment,the novel B1 receptor peptide antagonists of the present inventioncomprise an amino acid sequence as shown in any one of SEQ ID NOS:15-54.

According to some embodiments of this invention, one or more, andpreferably between one to nine amino acid residues, independentlyselected from any of the twenty genetically coded L-amino acids or thestereoisomeric D-amino acids, will be coupled to either or both ends ofthe peptide sequences as shown in SEQ ID NOS: 15-54.

In another embodiment, the present invention also provides conjugatedpeptides which have demonstrably superior pharmacokinetic properties invivo as compared to known peptide B1 antagonists yet they sufficientlybind to and antagonize the activity of the B1 receptor such that theymay be used therapeutically.

One aspect of the invention comprises a conjugated peptide of formula I:F—[(X¹)—(Y¹)_(n)]  Iwherein:

-   -   F is a vehicle covalently bound to X¹ or Y¹ (preferably F is a        PEG moiety or a derivative thereof);    -   X¹ and Y¹ are independently in each instance peptides of the        formula -L¹-P¹ and -L²-P², respectively;    -   L¹ and L² are independently in each instance linkers;    -   n is 0 to 3; and    -   P¹ and P² are independently in each instance peptide antagonists        of the bradykinin B1 receptor. Preferably, P¹ and P² comprise an        amino acid sequence as shown in any one of SEQ ID NOS: 5-26,        43-60, and derivatives thereof.

Another object of the present invention is to provide a pharmaceuticalcomposition comprising excipient carrier materials having a conjugatedpeptide of the invention dispersed therein.

Another object of the present invention is to provide therapeuticmethods of treatment which comprise administration to a mammal in needthereof a pharmaceutically effective amount of a composition comprisingexcipients and at least one peptide and/or conjugated peptide of theinvention.

The peptides and/or conjugated peptides of the invention havetherapeutic value for the treatment of diseases mediated by B1activation, including, but not limited to, inflammation and chronic painstates of inflammatory and neuropathic origin, septic shock, arthritis,osteoarthritis, angina, asthma, allergic rhinitis, and migraine.

The peptides and/or conjugated peptides of the invention may be used fortherapeutic or prophylactic purposes by formulating them withappropriate pharmaceutical carrier materials and administering aneffective amount to a patient, such as a human (or other mammal) in needthereof.

Additional useful peptides and/or conjugated peptides may result fromconservative modifications of the amino acid sequences of the peptidesand/or vehicle-conjugated peptides disclosed herein. Conservativemodifications will produce peptides and/or conjugated peptides havingfunctional, physical, and chemical characteristics similar to those ofthe peptides and/or conjugated peptide from which such modifications aremade. Such conservatively modified forms of the peptides and/orconjugated peptides disclosed herein are also contemplated as being anembodiment of the present invention.

Another aspect of the invention relates to a method of making aconjugated peptide as described herein, comprising the steps of:

-   -   -   reacting a compound having the structure:            (X¹)—(Y¹)_(N)            wherein:

    -   X¹ and Y¹ are independently in each instance peptides of the        formula -L¹-P¹ and -L²-P², respectively;

    -   L¹ and L² are independently in each instance linkers;

    -   n is 0 to 3; and

    -   P¹ and P² are independently in each instance peptide antagonists        of the bradykinin B1 receptor,

    -   with a vehicle (F) to give a conjugated peptide of the formula:        F—[(X¹)], F—[(X¹)]—F, F—[(X¹)—(Y¹)_(n)], F—(X¹ )—(Y¹)_(n), or        F—(X¹)—(Y¹)_(n)—F. Preferably F is a PEG moiety or a derivative        thereof. More preferably, P¹ and P² are independently in each        instance peptide antagonists of the bradykinin B1 receptor        comprising at least one of the peptide sequences shown in SEQ ID        NOS: 5-60. Even more preferably, X¹ is a peptide as shown in SEQ        ID NOS: 27-41. Additional aspects and advantages of the present        invention will become apparent upon consideration of the        detailed description of the invention which follows.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon the surprising finding that a classof peptides generally considered to be quite intolerant to substitutioncan be amino acid substituted at the N-terminus and/or conjugated tovarious vehicles at the N-terminus to provide therapeutically usefulpeptides and/or peptide conjugates with dramatically sustained efficacyprofiles as compared to the known peptides of the same class, andtherefore they allow for their use to manage inflammation and pain Thus,the peptides and/or peptide conjugates of the present invention providetremendous therapeutic advantage over known B1 peptide antagonists. Moreparticularly, the inventors have found that the previously describedshortcomings in known B1 peptide antagonists with respect to theirtherapeautic use are surmountable by substituting amino acids at theN-terminus and/or conjugating the peptide antagonists to vehicles suchas, but not limited to, polyethylene glycol (PEG) molecules usingpeptidyl or non-peptidyl linkers of defined size and composition whichmaximize preservation of antagonist activity and specificity whileprolonging efficacious half-life in vivo. Additionally (oralternatively), it was discovered that despite the slightly reduced invitro activity conferred to B1 peptide antagonists conjugated to largerPEG polymers, the extended circulating half-lives of the largePEG-conjugates provided significantly greater exposure and prolongedefficacy in vivo when compared to peptide conjugates conjugated tosmaller PEG polymers. In addition, the present inventors have found thatthe size of the PEG molecule attached to a peptide antagonist of the B1receptor is a critical parameter in optimizing the intrinsic antagonistactivity and the efficacious half-life in vivo. For example, anacetylated peptide B1 antagonist demonstrated efficacy in relevant invivo models of pain for a maximum of 4 hours following multiple dosing.Surprisingly, the same peptide conjugated to a 5 kD and a 20 kD PEGmolecule in the manner disclosed herein demonstrated efficacy for up to2 days and for at least 4 days, respectively, after a single bolusinjection.

Before the peptide and vehicle- or PEG-conjugated peptide antagonists ofthe bradykinin B1 receptor of the present invention and methods formaking and using such are described, it is to be understood that thisinvention is not limited to the particular peptides and/or conjugatedpeptide antagonists described, since peptides and/or conjugated peptideantagonists and methodologies contemplated by the present invention may,of course, vary slightly. It is to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting since the scope of the presentinvention will be limited only by the appended claims.

Bradykinin B1 receptor binding peptides contemplated for conjugation toa vehicle for purposes and in the manner as described herein include,but are not limited to, the novel B1 binding peptide antagonistsdisclosed herein as well as B1 binding peptide antagonists known in theart including, but not limited, to any peptide disclosed in any one ofthe following publications (each of which is hereby incorporated byreference in its entirety): Regoli et al., Bradykinin receptors andtheir antagonists. Eur. J. of Pharma., 348:1-10 (1998); Neugebauer, W.,et al., Kinin B₁ receptor antagonists with multi-enzymatic resistanceproperties. Can. J. Physiol. Pharmacol., 80:287-292 (2002); Stewart, J.M., et al, Bradykinin antagonists: present progress and futureprospects. Immunopharmacology, 43:155-161 (1999); Stewart, J. M., etal., Metabolism-Resistant Bradykinin Antagonists: Development andApplications. Biol. Chem., 382:37-41 (2001); PCT Publication WO98/07746; and U.S. Pat. Nos. 4,693,993, 4,801,613, 4,923,963, 5,648,336,5,834,431, 5,849,863, 5,935,932, 5,648,333, 5,385,889, 5,444,048, and5,541,286.

The terms used throughout this specification are defined as follows,unless otherwise limited in specific instances.

Natural amino acid residues are discussed in three ways: full name ofthe amino acid, standard three-letter code, or standard single-lettercode in accordance with the chart shown below. A = Ala G = Gly M = Met S= Ser C = Cys H = His N = Asn T = Thr D = Asp I = Ile P = Pro V = Val E= Glu K = Lys Q = Gln W = Trp F = Phe L = Leu R = Arg Y = Tyr

Unless clearly indicated otherwise, a designation herein of a natural ornon-natural amino acid is intended to encompass both the D- and L-isomerof the amino acid. Abbreviations used herein for unnatural amino acidsare the same as described in U.S. Pat. No. 5,834,431 , PCT publicationWO 98/07746, and Neugebauer, et al. (2002), each of which is herebyincorporated by reference in its entirety. Additionally, theabbreviation “Dab” and “D-Dab” is intended to refer to the L- andD-isomer of the unnatural amino acid, D-2-aminobutyric acid,respectively. The abbreviation “3′PaI” and “D-3′PaI” is intended torefer to the L- and D-isomer of the unnatural amino acid3′-pyridylalanine, respectively. Also, the abbreviation “Ig1” isintended to include both “Ig1a” and “Ig1b” (α-(1-indanyl)glycine andα-(2-indanyl)glycine, respectively). Similarly, “DIg1” is intended toinclude both “D-Ig1a” and “D-Ig1b” (the D-isomers ofα-(1-indanyl)glycine and α-(2-indanyl)glycine, respectively).Preferably, when used herein, Ig1 is Ig1b and D-Ig1 is D-Ig1b.

By “vehicle-conjugated peptide” or “conjugated peptide” is meant acompound which has biological activity and which when administered to amammal provides a therapeutic effect. The two parts include (1) at leastone B1 peptide antagonist and (2) at least one vehicle as definedhereinbelow covalently bound to a residue of the peptide itself or to apeptidyl or non-peptidyl linker (including but not limited to aromaticlinkers) that is covalently bound to a residue of the peptide.

By “PEG-conjugated peptide” or “PEGylated peptide” is meant a two partcompound which has biological activity and which when administered to amammal provides a therapeutic effect. The two parts include (1) at leastone B1 peptide antagonist and (2) at least one polyethylene glycol (PEG)moiety covalently bound to a residue of the peptide itself or to apeptidyl or non-peptidyl linker (including but not limited to aromaticlinkers) that is covalently bound to a residue of the peptide.

By “polyethylene glycol” or “PEG” is meant a polyalkylene glycolcompound or a derivative thereof, with or without coupling agents orderivatization with coupling or activating moieties (e.g., with thiol,triflate, tresylate, azirdine, oxirane, orthopyridyl disulphide,vinylsulfone, iodoacetamide or a maleimide moiety).

PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods well known in the art (Sandler and Karo,Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). Inthe present application, the term “PEG” is used broadly to encompass anypolyethylene glycol molecule, in mono- to poly functional form, withoutregard to size or to modification at an end of the PEG, and can berepresented by the formula:X—O(CH₂CH₂O)_(n-1)CH₂CH₂OH,   IIwhere n is 20 to 2300 and X is H or a terminal modification, e.g., aC₁₋₄ alkyl.

Preferably, a PEG used in the invention terminates on one end withhydroxy or methoxy, i.e., X is H or CH₃ (“methoxy PEG”). It is notedthat when X═CH₃, the other end of the PEG, which is shown in formula IIterminating in OH, covalently attaches to an activating moiety via anether oxygen bond. When X═H, both ends of the PEG are attached toactivating moieties via ether bonds giving rise to linearbis-functionalized PEGs. When used in a chemical structure, the term“PEG” includes the formula II above without the hydrogen of the hydroxylgroup shown, leaving the oxygen available to react with a free carbonatom of a linker to form an ether bond. More specifically, in order toconjugate PEG to a peptide, PEG must be in an “activated” form.Activated PEG can be represented by formula III:(PEG)-(A)   IIIwhere PEG (defined supra.) covalently attaches to a carbon atom of theactivation moiety (A) to form an ether bond, and (A) contains a reactivegroup which can react with an amino, imino, or thiol group on an aminoacid residue of a peptide or a linker moiety covalently attached to thepeptide.

Methods for the preparation of activated PEGs are well known in the art,e.g., see U.S. Pat. Nos. 5,643,575, 5,919,455, 5,932,462, and PCTpublication WO 95/06058 (each of which is hereby incorporated byreference in their entirety). Suitable activated PEGs can be produced bya number of conventional reactions. For example, an N-hydroxysuccinimideester of a PEG (M-NHS-PEG) can be prepared from PEG-monomethyl ether(which is commercially available from Union Carbide) by reaction withN,N′-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS),according to the method of Buckmann and Merr, Makromol. Chem.,182:1379-1384 (1981). Other activated PEGs, such as PEG-aldehydes, canbe obtained from a commercial source, e.g., Nektar Therapeutics(Huntsville, Ala.) or Enzon, Inc. (Piscataway, N.J.). Examples ofpreferred activated PEG for purposes of the present invention arePEG-propionaldehyde and PEG-butyraldehyde which are commerciallyavailable from Nektar Therapeutics (Huntsville, Ala.).PEG-propionaldehyde is represented by the formula PEG-CH₂CH₂CHO and isdescribed in U.S. Pat. No. 5,252,714, which is entirely incorporated byreference herein. In addition, bifunctional PEG aldehydes may be used toprepare dimeric conjugates.

Additional preferred amine reactive PEGs include: methoxy-PEGsuccinimidyl propionate (mPEG-SPA) and methoxy-PEG succinimido butanoatemPEG-SBA), mPEG-benzotriazole carbonate or mPEG-p-nitrophenyl carbonatewhich are available in a variety of molecular weights from NektarTherapeutics (Huntsville, Ala.), Enzon, Inc. (Piscataway, N.J.), or NOFCorporation (Tokyo, Japan). Additional preferred activated PEG moietiesinclude thiol reactive functionalities including, but not limited to,PEG vinyl sulfones, represented by the formula PEG-CH₂CH₂SO₂—CH═CH₂,mPEG-iodoacetate and mPEG-thioesters depicted below:

Another preferred activated PEG for generating the PEG-conjugatedpeptides of the present invention is PEG-maleimide. Compounds such asmaleimido monomethoxy PEGs are particularly useful for generating thePEG-conjugated peptides of the invention.

An even more preferred activated PEG for generating the PEG-conjugatedpeptides of the present invention is a multivalent PEG having more thanone activated residues. Preferred multivalent PEG moieties include, butare not limited to, those shown below:

Any molecular mass for a PEG can be used as practically desired, e.g.,from about 1,000 Daltons (Da) to 100,000 Da (n is 20 to 2300). Thenumber of repeating units “n” in the PEG is approximated for themolecular mass described in Daltons. It is preferred that the combinedmolecular mass of PEG on an activated linker is suitable forpharmaceutical use. Thus, the combined molecular mass of the PEGmolecules should not exceed 100,000 Da.

Preferably, the combined or total molecular mass of PEG used in aPEG-conjugated peptide of the present invention is from about 3,000 Dato 60,000 Da (total n is from 70 to 1,400), more preferably from about8,800 Da to 36,000 Da (total n is about 200 to about 820). The mostpreferred combined mass for PEG is from about 20,000 Da to 24,000 Da(total n is about 450 to about 540).

Other polyalkylene glycol compounds, such as polypropylene glycol, maybe used in the present invention. Other appropriate polyalkylene glycolcompounds include, but are not limited to, charged or neutral polymersof the following types: dextran, colominic acids or other carbohydratebased polymers, polymers of amino acids, and biotin derivatives.

The term “comprising” means that a peptide or conjugated peptide mayinclude additional molecular entities, including, but not limited to,amino acids, on either or both of the N- or C-termini of the givensequence. Of course, these additional molecular entities should notsignificantly interfere with the activity of the peptide or conjugatedpeptide.

As used herein, the term “native peptide” refers to an unconjugated B1peptide antagonist disclosed herein or known in the art.

The terms “derivatizing” and “derivative” or “derivatized” compriseprocesses and resulting peptides or conjugated peptides, respectively,in which (1) the peptide or conjugated peptide has a cyclic portion; forexample, cross-linking between cysteinyl residues within the conjugatedpeptide; (2) the peptide or conjugated peptide is cross-linked or has across-linking site; for example, the peptide or conjugated peptide has acysteinyl residue and thus forms cross-linked dimers in culture or invivo; (3) the N-terminus of a conjugated peptide having a —NH₂ terminalgroup is replaced by —NRR¹, NRC(O)R¹, —NRC(O)OR¹, —NRS(O)₂R¹,—NHC(O)NHR, a succinimide group, or substituted or unsubstitutedbenzyloxycarbonyl-NH—, wherein R and R¹ and the ring substituents are asdefined hereinafter; (5) the C-terminus is replaced by —C(O)R² or —NR³R⁴wherein R², R³ and R⁴ are as defined hereinafter; and (6) conjugatedpeptides in which individual amino acid moieties are modified throughtreatment with agents capable of reacting with selected side chains orterminal residues. Derivatives are further described hereinafter.

The term “B1” means the bradykinin B1 receptor (see, Judith M Hall, Areview of BK receptors. Pharmac. Ther. 56:131-190 (1992)). Unlessspecifically noted otherwise, B1 or bradykinin B1 receptor is intendedto mean the human bradykinin B1 receptor (hB1). Preferably, hB1 is thewild-type receptor. More preferably, hB1 is the bradykinin receptordescribed in GenBank Accession no. AJ238044.

The term “peptide” as used generally herein refers to molecules of 4 to40 amino acids, with molecules of 10 to 20 amino acids being preferredand those of 15 to 18 amino acids being most preferred. The term“di-peptide” as used herein refers to a molecule of two amino acids. Theterm “tri-peptide” as used herein refers to a molecule of three aminoacids.

Structural analysis of protein-protein interaction may also be used tosuggest peptides that mimic the binding activity of large proteinligands. In such an analysis, the crystal structure may suggest theidentity and relative orientation of critical residues of the proteinligand from which an analogous peptide may be designed. See, forexample, Takasaki et al., Nature Biotech., Volume 15, pages 1266-1270(1997). These analytical methods may also be used to investigate theinteraction between a receptor protein and a peptide, vehicle-conjugatedpeptide, or PEG-conjugated peptide of the present invention, which maysuggest further modification of the peptide or peptide conjugates toincrease binding affinity.

As used herein, the terms “effective amount” and “therapeuticallyeffective amount” when used with reference to a peptide,vehicle-conjugated peptide, or PEG-conjugated peptide B1 antagonistrefers to an amount or dosage sufficient to produce a desired result(i.e., for therapy with the peptides, vehicle-conjugated peptides,and/or PEG-conjugated peptide B1 antagonists of the present invention.In the context of the present invention, the desired result is a desiredreduction in inflammation and/or pain, for example, or to support anobservable decrease in the level of one or more biological activities ofB1. More specifically, a therapeutically effective amount is an amountof the peptide and/or conjugated peptide sufficient to inhibit, for someperiod of time, one or more of the clinically defined pathologicalprocesses associated with the condition at issue, e.g., inflammation orpain, in a subject treated in vivo with the agent(s). The effectiveamount may vary depending on the specific peptide and/or conjugatedpeptide B1 antagonist selected, and is also dependent on a variety offactors and conditions related to the subject to be treated and theseverity of the disorder. For example, if the peptide and/or conjugatedpeptide B1 antagonist is to be administered in vivo, factors such as theage, weight and health of the patient as well as dose response curvesand toxicity data obtained in preclinical animal work would be amongthose considered. If the agent(s) is to be contacted with the cells invitro, one would also design a variety of pre-clinical in vitro studiesto assess such parameters as uptake, half-life, dose, toxicity, etc. Thedetermination of an effective amount or a therapeutically effectiveamount for a given agent is well within the ability of those skilled inthe art.

The term “pharmacologically active” means that a substance so describedis determined to have activity that affects a medical parameter ordisease state (for example, pain). In the context of the invention, thisterm typically refers to a B1-induced or B1-mediated disease or abnormalmedical condition or disorder, and more specifically, to antagonism ofinflammation or pain.

The terms “antagonist”, “inhibitor”, and “inverse agonist” (e.g., see,Rianne A. F. de Ligt, et. al, British Journal of Pharmacology 2000, 130,131) refer to a molecule that blocks, impedes, reduces, lessens or insome way interferes with the biological activity of the associatedprotein of interest. A preferred “antagonist” or “inhibitor” of thepresent invention is a molecule that binds to and inhibits B1 with anIC₅₀ of 500 nM or less in in vitro assays of B1 activity. A morepreferred “antagonist” or “inhibitor” of the present invention is amolecule that binds to and inhibits B1 with an IC₅₀ of 100 nM or less inin vitro assays of B1 activity. A most preferred “antagonist” or“inhibitor” of the present invention is a molecule that binds to andinhibits B1 with an IC₅₀ of 50 nM or less in in vitro assays of B1activity and prevents, ameliorates or abolishes pain as measured in atleast one generally accepted in vivo animal model of pain and/orinhibits biochemical challenges in in vivo animal models of edema,inflammation, or pain.

Additionally, physiologically acceptable salts of the peptides orconjugated peptides of the invention are also encompassed herein. Thephrases “physiologically acceptable salts” and “pharmacologicallyacceptable salts” as used herein are interchangeable are intended toinclude any salts that are known or later discovered to bepharmaceutically acceptable (i.e., useful in the treatment of awarm-blooded animal). Some specific examples are: acetate;trifluoroacetate; hydrohalides, such as hydrochloride and hydrobromide;sulfate; citrate; tartrate; glycolate; oxalate; salts of inorganic andorganic acids, including, but not limited to, hydrochloric acid,hydrobromic acid, sulfuric acid, phosphoric acid, methanesulphonic acid,ethanesulfonic acid, malic acid, acetic acid, oxalic acid, tartaricacid, citric acid, lactic acid, fumaric acid, succinic acid, maleicacid, salicylic acid, benzoic acid, phenylacetic acid, mandelic acid andthe like. When compounds of the invention include an acidic functionsuch as a carboxy group, then suitable pharmaceutically acceptablecation pairs for the carboxy group are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium, quaternaryammonium cations and the like. For additional examples of“pharmacologically acceptable salts,” see infra and Berge et al., J.Pharm. Sci. 66:1 (1977).

“Protecting group” generally refers to groups well known in the artwhich are used to prevent selected reactive groups, such as carboxy,amino, hydroxy, mercapto and the like, from undergoing undesiredreactions, such as nucleophilic, electrophilic, oxidation, reduction andthe like. Preferred protecting groups are indicated herein whereappropriate. Examples of amino protecting groups include, but are notlimited to, aralkyl, substituted aralkyl, cycloalkenylalkyl andsubstituted cycloalkenyl alkyl, allyl, substituted allyl, acyl,alkoxycarbonyl, aralkoxycarbonyl, silyl and the like. Examples ofaralkyl include, but are not limited to, benzyl, ortho-methylbenzyl,trityl and benzhydryl, which can be optionally substituted with halogen,alkyl, alkoxy, hydroxy, nitro, acylamino, acyl and the like, and salts,such as phosphonium and ammonium salts. Examples of aryl groups includephenyl, naphthyl, indanyl, anthracenyl, 9-(9-phenylfluorenyl),phenanthrenyl, durenyl and the like. Examples of cycloalkenylalkyl orsubstituted cycloalkylenylalkyl radicals, preferably have 6-10 carbonatoms, include, but are not limited to, cyclohexenyl methyl and thelike. Suitable acyl, alkoxycarbonyl and aralkoxycarbonyl groups includebenzyloxycarbonyl, t-butoxycarbonyl, iso-butoxycarbonyl, benzoyl,substituted benzoyl, butyryl, acetyl, tri-fluoroacetyl, tri-chloroacetyl, phthaloyl and the like. A mixture of protecting groups can beused to protect the same amino group, such as a primary amino group canbe protected by both an aralkyl group and an aralkoxycarbonyl group.Amino protecting groups can also form a heterocyclic ring with thenitrogen to which they are attached, for example,1,2-bis(methylene)benzene, phthalimidyl, succinimidyl, maleimidyl andthe like and where these heterocyclic groups can further includeadjoining aryl and cycloalkyl rings. In addition, the heterocyclicgroups can be mono-, di- or tri-substituted, such as nitrophthalimidyl.Amino groups may also be protected against undesired reactions, such asoxidation, through the formation of an addition salt, such ashydrochloride, toluenesulfonic acid, trifluoroacetic acid and the like.Many of the amino protecting groups are also suitable for protectingcarboxy, hydroxy and mercapto groups. For example, aralkyl groups. Alkylgroups are also suitable groups for protecting hydroxy and mercaptogroups, such as tert-butyl.

Silyl protecting groups are silicon atoms optionally substituted by oneor more alkyl, aryl and aralkyl groups. Suitable silyl protecting groupsinclude, but are not limited to, trimethylsilyl, triethylsilyl,tri-isopropylsilyl, tert-butyldimethylsilyl, dimethylphenylsilyl,1,2-bis(dimethylsilyl)benzene, 1,2-bis(dimethylsilyl)ethane anddiphenylmethylsilyl. Silylation of an amino groups provide mono- ordi-silylamino groups. Silylation of aminoalcohol compounds can lead to aN,N,O-tri-silyl derivative. Removal of the silyl function from a silylether function is readily accomplished by treatment with, for example, ametal hydroxide or ammonium fluoride reagent, either as a discretereaction step or in situ during a reaction with the alcohol group.Suitable silylating agents are, for example, trimethylsilyl chloride,tert-butyl-dimethylsilyl chloride, phenyldimethylsilyl chloride,diphenylmethyl silyl chloride or their combination products withimidazole or DMF. Methods for silylation of amines and removal of silylprotecting groups are well known to those skilled in the art. Methods ofpreparation of these amine derivatives from corresponding amino acids,amino acid amides or amino acid esters are also well known to thoseskilled in the art of organic chemistry including amino acid/amino acidester or aminoalcohol chemistry.

Protecting groups are removed under conditions which will not affect theremaining portion of the molecule. These methods are well known in theart and include acid hydrolysis, hydrogenolysis and the like. Apreferred method involves removal of a protecting group, such as removalof a benzyloxycarbonyl group by hydrogenolysis utilizing palladium oncarbon in a suitable solvent system such as an alcohol, acetic acid, andthe like or mixtures thereof. A t-butoxy-carbonyl protecting group canbe removed utilizing an inorganic or organic acid, such as HCl ortrifluoroacetic acid, in a suitable solvent system, such as dioxane ormethylene chloride. The resulting amino salt can readily be neutralizedto yield the free amine. Carboxy protecting group, such as methyl,ethyl, benzyl, tert-butyl, 4-methoxyphenylmethyl and the like, can beremoved under hydrolysis and hydrogenolysis conditions well known tothose skilled in the art.

It should be noted that compounds of the invention may contain groupsthat may exist in tautomeric forms, such as cyclic and acyclic amidineand guanidine groups, heteroatom substituted heteroaryl groups (Y′═O, S,NR), and the like, which are illustrated in the following examples:

and though one form is named, described, displayed and/or claimedherein, all the tautomeric forms are intended to be inherently includedin such name, description, display and/or claim.

Prodrugs of the compounds of this invention are also contemplated bythis invention. A prodrug is an active or inactive compound that ismodified chemically through in vivo physiological action, such ashydrolysis, metabolism and the like, into a compound of this inventionfollowing administration of the prodrug to a patient. The suitabilityand techniques involved in making and using prodrugs are well known bythose skilled in the art. For a general discussion of prodrugs involvingesters see Svensson and Tunek Drug Metabolism Reviews 165 (1988) andBundgaard Design of Prodrugs, Elsevier (1985). Examples of a maskedcarboxylate anion include a variety of esters, such as alkyl (forexample, methyl, ethyl), cycloalkyl (for example, cyclohexyl), aralkyl(for example, benzyl, p-methoxybenzyl), and alkylcarbonyloxyalkyl (forexample, pivaloyloxymethyl). Amines have been masked asarylcarbonyloxymethyl substituted derivatives which are cleaved byesterases in vivo releasing the free drug and formaldehyde (Bundgaard J.Med. Chem. 2503 (1989)). Also, drugs containing an acidic NH group, suchas imidazole, imide, indole and the like, have been masked withN-acyloxymethyl groups (Bundgaard Design of Prodrugs, Elsevier (1985)).Hydroxy groups have been masked as esters and ethers. EP 039,051 (Sloanand Little, Apr. 11, 1981) discloses Mannich-base hydroxamic acidprodrugs, their preparation and use.

Structure of Conjugated Peptides

In General. The vehicle-conjugated peptides of the present invention maybe described by the following formula:F—[(X¹)—(Y¹)_(n)]  (IV)wherein:

-   -   X¹ and Y¹ are independently in each instance peptides of the        formula -L′-P¹ and -L²-P², respectively;    -   F is a vehicle covalently bound to X¹ or Y¹;    -   L¹ and L² are independently in each instance absent or linkers        having from 0 to 9 amino acid residues;    -   n is 0 to 3; and    -   P¹ and, if present, P² are independently in each instance        peptide antagonists of the bradykinin B1 receptor.

The vehicle-conjugated peptides of formula IV will comprise preferredembodiments wherein P¹ and, if present, p² are independently in eachinstance peptide antagonists of the bradykinin B1 receptor having apeptide sequence as shown in any one of SEQ ID NOS: 5-60 and derivativesthereof.

Additional preferred embodiments of the vehicle-conjugated peptides willinclude vehicle-conjugated peptides of formula IV wherein P¹ and, ifpresent, P² is defined by the formula:NH₂-a⁰a¹a²a³a⁴a⁵a⁶a⁷a⁸a⁹a¹⁰a¹¹a¹²a¹³a¹⁴-COOHwherein:

-   -   a⁰ is a basic or neutral aromatic, aliphatic, heterocyclic, or        alicyclic amino acid, di-peptide or tri-peptide containing        either one or two residues having basic side chains, or absent;    -   a¹, a², a^(3,) and a⁴ are independently in each instance basic        or neutral aromatic, aliphatic, heterocyclic, or alicyclic amino        acids;    -   a⁶ is Ser;    -   a⁵, a⁷, and a⁸ are independently in each instance aromatic,        aliphatic, heterocyclic, or alicyclic amino acids, provided that        at least one of a⁵, a⁷ ₁ and a⁸ is selected from Chg, Cpg, Ig1a,        Ig1b, Niga and Nigb of the D- or L-configuration; and    -   a⁹, a¹⁰, a¹¹, a¹², a¹³, and a¹⁴ are independently in each        instance any natural amino acid or absent.

More preferably, P¹ and, if present, P² are defined by the formula:NH₂-a⁰a¹a²a³a⁴a⁵a⁶a⁷a⁸a⁹a¹⁰a¹¹a¹²a¹³a¹⁴-COOHwherein:

-   -   a⁰ is a basic amino acid, a di-peptide containing either one or        two residue with basic side chains, or absent;    -   a¹ is a basic amino acid;    -   a² is Pro;    -   a³ is Hyp;    -   a⁴ is Gly;    -   a⁵ and a⁸ is an Indanyl amino acid;    -   a⁶ is Ser;    -   a⁷ is a D-Indanyl amino acid;    -   a⁸is Cpg; and    -   a⁹, a¹⁰, a¹¹, a¹², a¹³, and a¹⁴ are independently in each        instance any natural amino acid or absent.

Even more preferably, P¹ and, if present, P² are defined by the formula:NH₂-a⁰a¹a²a³a⁴a⁵ a⁶a⁷a⁸a⁹a¹⁰a¹¹a¹²a¹³a¹⁴-COOHwherein:

-   -   a⁰ is a basic amino acid, di-peptide containing either one or        two basic side chains, or absent;    -   a¹ is a basic amino acid;    -   a² is Pro;    -   a³ is Hyp;    -   a⁴ is Gly;    -   a⁵ is Cpg;    -   a⁶ is Ser;    -   a⁷ is DTic;    -   a⁸ is Cpg; and    -   a⁹, a¹⁰, a¹¹, a¹², a¹³, and a¹⁴ are independently in each        instance any natural amino acid or absent.

Even more preferably, vehicle-conjugated peptides of the presentinvention include vehicle-conjugated peptides of formula IV wherein n=0and X¹ is a peptide selected from the group consisting of peptides asshown in SEQ ID NOS: 27-41 and derivatives thereof.

The present invention also provides PEG-conjugated peptides which bindto and antagonize the activity of bradykinin B1 receptors (B1) and whichhave demonstrably superior pharmacokinetic properties in vivo ascompared to unconjugated peptide B1 antagonists. The PEG-conjugatedpeptides of the present invention may be described by the followingformula (V):F—[(X¹)—(Y¹)_(n)]  Vwherein:

-   -   X¹ and Y¹ are independently in each instance peptides of the        formula -L′-P¹ and -L²-P², respectively;    -   F is a PEG moiety covalently bound to X¹ or Y¹;    -   L¹ and L² are independently in each instance absent or linkers        having from 0 to 9 amino acid residues;    -   n is 0 to 3; and    -   P¹ and, if present, P² are independently in each instance        peptide antagonists of the bradykinin B1 receptor.

The PEG-conjugated peptides of formula V will comprise preferredembodiments wherein P¹ and, if present, P² are independently in eachinstance peptide antagonists of the bradykinin B1 receptor having apeptide sequence as shown in any one of SEQ ID NOS: 5-60 and derivativesthereof.

Additional preferred embodiments of the PEG-conjugated peptides willinclude PEG-conjugates of formula V wherein P¹ and, if present, P²isdefined by the formula:NH₂-a⁰a¹a²a³a⁴a⁵a⁶a⁷a⁸a⁹a¹⁰a¹¹a¹²a¹³a¹⁴-COOHwherein:

-   -   a⁰ is a basic or neutral aromatic, aliphatic, heterocyclic, or        alicyclic amino acid, basic di-peptide or tri-peptide, or        absent;    -   a¹, a², a^(3,) and a⁴ are independently in each instance basic        or neutral aromatic, aliphatic, heterocyclic, or alicyclic amino        acids;    -   a⁶is Ser;    -   a⁵, a⁷, and a⁸ are aromatic, aliphatic, heterocyclic, or        alicyclic amino acids, provided that at least one of a⁵, a⁷, and        a⁸ is selected from Chg, Cpg, Ig1a, Ig1b, Niga and Nigb of the        D- or L-configuration; and    -   a⁹, a¹⁰, a¹¹, a¹², a¹³, and a¹⁴ are independently in each        instance any natural amino acid or absent.

More preferably, P¹ and, if present, P² are defined by the formula:NH₂-a⁰a¹a²a³a⁴a⁵a⁶ ^(a) ⁷a⁸a⁹a¹⁰a¹¹a¹²a¹³a¹⁴-COOHwherein:

-   -   a⁰ is a basic amino acid, a basic di-peptide or absent;    -   a¹ is a basic amino acid;    -   a² is Pro;    -   a³ is Hyp;    -   a⁴ is Gly;    -   a⁵ and a⁸ is an Indanyl amino acid;    -   a⁶ is Ser;    -   a⁷ is a D-Indanyl amino acid;    -   a⁸ is Cpg; and    -   a⁹, a¹⁰, a¹¹, a¹², a¹³, and a¹⁴ are independently in each        instance any natural amino acid or absent.

Even more preferably, P¹ and, if present, P² are defined by the formula:NH₂-a⁰a¹a²a³a⁴a⁵a⁶a⁷a⁸a⁹a¹⁰a¹¹a¹²a¹³a¹⁴-COOHwherein:

-   -   a⁰ is a basic amino acid, a basic di-peptide, or absent;    -   a¹ is a basic amino acid;    -   a² is Pro;    -   a³ is Hyp;    -   a⁴ is Gly;    -   a⁵ is Cpg;    -   a⁶ is Ser;    -   a⁷ is DTic;    -   a⁸ is Cpg; and    -   a⁹, a¹⁰, a¹¹, a¹², a¹³, and a¹⁴ are independently in each        instance any natural amino acid or independently absent.

Even more preferred PEG-conjugated peptides of the present inventioninclude PEG-conjugated peptides of formula V wherein n=0 and X¹ is apeptide selected from the group consisting of peptides having an aminoacid sequence as shown in SEQ ID NOS: 27-41 and derivatives thereof.Even more preferred PEG-conjugated peptides of the present inventioninclude wherein n=0 and X¹ is a peptide selected from the groupconsisting of peptides having an amino acid sequence as shown in SEQ IDNOS: 27-41 and derivatives thereof. Even more preferably, thePEG-conjugated peptides of the present invention may be described by thefollowing formula:F′—R_(z),   VIor a physiologically acceptable salt thereof, wherein:

-   -   F′ is a multivalent vehicle;    -   R is independently in each instance —(X¹)—(Y¹)_(n) wherein R is        covalently bound to F′;    -   X¹ and Y¹ are independently in each instance peptides of the        formula -L¹-P¹ and -L²-P², respectively;    -   L¹ and L² are independently in each instance absent or linkers        having from 0 to 9 amino acid residues;    -   n is 0 to 3;    -   Z is 2 to 8; and    -   P¹ and P² are independently in each instance peptide antagonists        of the bradykinin B1 receptor.

Even more preferred PEG-conjugated peptides of the present inventioninclude PEG-conjugated peptides of formula VI wherein n is 0, Z is 4 to8, and X¹ is a peptide selected from the group consisting of peptideshaving an amino acid sequence as shown in SEQ ID NOS: 27-41 andderivatives thereof.

Also intended as part of the present invention are peptide conjugateshaving peptide sequences that are fragments (i.e., “subsequences”),analogs, and derivatives of P¹ and, if present, P² as defined herein andwherein such conjugated peptides are substantially equivalent withrespect to in vitro and/or in vivo anti-B1 activity as the peptideconjugates specifically disclosed herein.

The term “analog” is intended to mean molecules representing one or moreamino acid substitutions, deletions and/or additions derived from thelinear array of amino acids of the peptides, conjugated-peptides(unconjugated P¹ and, if present, P²), and/or any peptidyl linker (L) ofthe vehicle- or PEG-conjugated peptides provided for by the formulas(IV) and (V), respectively, and which result in molecules which aresubstantially equivalent with respect to in vitro and/or in vivo anti-B1activity as compared to an analogous unconjugated peptide or conjugatedpeptide specifically disclosed herein.

The conjugated peptide analogs in accordance with this invention willtypically have one or more amino acid substitutions, deletions and/orinsertions in the sequence of (P) (P¹ and/or, if present, P²) or (L) (L¹and/or, if present, L²). It is generally recognized that conservativeamino acid changes are least likely to perturb the structure and/orfunction of a polypeptide and generally involve substitution of oneamino acid with another that is similar in structure and/or function(e.g., amino acids with side chains similar in size, charge and/orshape). The nature of these substitutions are well known to one skilledin the art and exemplary amino acid substitutions are summarized inTables 1 and 2. TABLE 1 Amino Acid Substitutions Basic: Arg; Lys; His;Acidic: Glu; Asp Polar: Glu; Asp; Gln; Asn; Ser; Thr Hydrophilic: Asp;Glu; Asn; Ser; Thr; Tyr Hydrophobia: Ala; Met; Ile; Leu; nor-Leu; ValAromatic: Phe; Trp; Tyr Small: Gly; Ala; Ser; Thr; Met

TABLE 2 Amino Acid Substitutions Most Amino Preferred Acid PreferredSubstitutions Substitution Ala Gly; Leu; Ile; Asn; Pro Val Arg Ala; Asn;Gln; Ser Lys Asn Arg; Gln; His; Lys; Ser; Tyr Gln Asp Asn; Ser; Thr; GlnGlu Cys Ala Ser Gln Ala; Arg; Glu; Leu; Lys; Met; Ser; Tyr Asn Glu Gln;Ser; Thr; Asn Asp Gly Pro His Asn; Gln; Lys; Tyr; Phe Arg Ile Tyr; Val;Met; Ala; Phe; nor-Leu Leu Leu nor-Leu; Ile; Val; Met; Ala; Phe Ile LysAsn; Asp; Ala; Glu; Gln; Ser; Tyr Arg Met Ala; Gln; Tyr; Trp; Phe LeuPhe Leu; Val; Ile; Ala; Met Leu Pro Ile; Val Gly Ser Ala; Asn; Asp; Gly;Lys Thr Thr Ala; Gly; Ile; Val; Lys Ser Trp Phe; Tyr; His Tyr Tyr Trp;Thr; Ser Phe Val Ala; Ile; Met; Phe; Tyr; nor-Leu LeuChanging from A, F, H, I, L, M, P, V, W, or Y to C is more preferred ifthe new cysteine remains as a free thiol.

Desired amino acid substitutions (whether conservative ornon-conservative) can be determined by those skilled in the art at thetime such substitutions are desired. For example, amino acidsubstitutions can be used to identify important residues of the peptidesequence, or to increase or decrease the affinity of the unconjugatedand/or conjugated peptide molecules described herein.

In certain embodiments, conservative amino acid substitutions alsoencompass non-naturally occurring amino acid residues which aretypically incorporated by chemical peptide synthesis.

As noted in the foregoing section, naturally occurring residues may bedivided into classes based on common side chain properties that may beuseful for modifications of sequence. For example, non-conservativesubstitutions may involve the exchange of a member of one of theseclasses for a member from another class. Such substituted residues maybe introduced into regions of the peptide that are homologous withnon-human orthologs, or into the non-homologous regions of the molecule.In addition, one may also make modifications using P or G for thepurpose of influencing chain orientation.

In making such modifications, the hydropathic index of amino acids maybe considered. Each amino acid has been assigned a hydropathic index onthe basis of their hydrophobicity and charge characteristics, these are:isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is understood in the art.Kyte et al., J. Mol. Biol., 157: 105-131 (1982). It is known thatcertain amino acids may be substituted for other amino acids having asimilar hydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, thesubstitution of amino acids whose hydropathic indices are within ±2 ispreferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. Thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with itsimmunogenicity and antigenicity, i.e., with a biological property of theprotein.

The following hydrophilicity values have been assigned to amino acidresidues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate(+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine(0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine(−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine(−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5);tryptophan (−3.4). In making changes based upon similar hydrophilicityvalues, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those which are within ±1 are particularlypreferred, and those within ±0.5 are even more particularly preferred.One may also identify epitopes from primary amino acid sequences on thebasis of hydrophilicity. These regions are also referred to as “epitopiccore regions.”

A skilled artisan will be able to determine suitable analogs of theunconjugated and/or conjugated peptides set forth herein using wellknown techniques. One skilled in the art would also know that one maysubstitute chemically similar amino acids for residues occurring in thenative peptide while retaining activity (conservative amino acid residuesubstitutions). Therefore, even areas that may be important forbiological activity or for structure may be subject to conservativeamino acid substitutions without destroying the biological activity orwithout adversely affecting the unconjugated peptide or conjugatedpeptide structure.

Additionally, one skilled in the art can review structure-functionstudies identifying residues within the unconjugated and/or conjugatedpeptide sequence that are important for activity or structure. In viewof such a comparison, one can predict the importance of amino acidresidues in a peptide sequence. One skilled in the art may opt tosubstitute chemically similar amino acid substitutions for suchpredicted important amino acid residues of unconjugated and/orconjugated peptides of the present invention.

A number of scientific publications have been devoted to the predictionof secondary structure. See Moult J., Curr. Op. in Biotech.,7(4):422-427 (1996), Chou et al., Biochemistry, 13(2):222-245 (1974);Chou et al., Biochemistry, 113(2):211-222 (1974); Chou et al., Adv.Enzymol. Relat. Areas Mol. Biol., 47:45-148 (1978); Chou et al., Ann.Rev. Biochem., 47:251-276 and Chou et al., Biophys. J., 26:367-384(1979). Moreover, computer programs are currently available to assistwith predicting secondary structure.

Additional methods of predicting secondary structure include “threading”(Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87 (1997); Sippl et al.,Structure, 4(1): 15-9 (1996)), “profile analysis” (Bowie et al.,Science, 253:164-170 (1991); Gribskov et al., Meth. Enzym., 183:146-159(1990); Gribskov et al., Proc. Nat. Acad. Sci., 84(13):4355-8 (1987)),and “evolutionary linkage” (See Home, supra, and Brenner, supra).

Peptide and/or conjugated peptide analogs and derivatives in accordancewith the invention will be useful for the same purposes for which theanalogous peptides and/or conjugated peptides specifically disclosedherein are useful (i.e., antagonists of B1 activity in vitro and/or invivo).

Peptides. Peptides of the present invention include peptides comprisingthe sequences shown in SEQ ID NOS: 15-35 and 39-54. The peptidesequences P¹ and, if present, P² (P) within the vehicle- orPEG-conjugated peptides of the present invention include, as mentioned,peptides that bind to and antagonize (e.g., decrease) the activity ofB1. Preferred vehicle- or PEG-conjugated peptides of the presentinvention comprise at least one peptide sequence selected from the groupconsisting of SEQ ID NOS: 5-60 and derivatives thereof. More preferably,vehicle- or PEG-conjugated peptides of the present invention comprise atleast one peptide sequence selected from the group consisting of SEQ IDNOS: 27-41 and derivatives thereof. TABLE 3 Bradykinin PeptidesReceptor/Effect Peptide Peptide Sequence B2/B1 Agonist Bradykinin, BK            Arg Pro Pro Gly Phe Ser Pro Phe Arg (SEQ ID NO:1) B2 AgonistKallidin, Lys-BK        Lys  Arg Pro Pro Gly Phe Ser Pro Phe Arg (SEQ IDNO:2) B2 Agonist Met-Lys-BK    Met Lys  Arg Pro Pro Gly Phe Ser Pro PheArg (SEQ ID NO:3) B1 Agonist des-Arg-BK             Arg Pro Pro Gly PheSer Pro Phe (SEQ ID NO:4) B1 Antagonist [Leu8]-Des-Arg9-BK            Arg Pro Pro Gly Phe Ser Pro Leu (SEQ ID NO:5) B1 AntagonistDALK        Lys  Arg Pro Pro Gly Phe Ser Pro Leu (SEQ ID NO:6) B2Antagonist (SEQ ID NO:7)        DArg Arg Pro Hyp Gly Thi Ser DTic OicArg B1/B2 (SEQ ID NO:8)        DArg Arg Pro Hyp Gly Thi Ser DTic OicAntagonist B2 Antagonist (SEQ ID NO:9)        DArg Arg Pro Hyp Gly ThiSer DHpe Oic Arg B1 (SEQ ID NO:10)                             Me-Antagonist Ac Lys Lys  Arg Pro Pro Gly Phe Ser D-β-NaI Ile B1/B2 (SEQ IDNO:11)        DArg Arg Pro Hyp Gly Igl Ser DIgl Oic Arg Antagonist B1Antagonist (SEQ ID NO:12)    Lys Lys  Arg Pro Hyp Gly Igl Ser DIgl OicB1 Antagonist (SEQ ID NO:13)    Lys Lys  Arg Pro Hyp Gly Cpg Ser DticCpg B1/B2 (SEQ ID NO:14)        DArg Arg Pro Hyp Gly Igl Ser Df5f IglArg Antagonist B1 Antagonist (SEQ ID NO:15)    DOrnLys  Arg Pro Hyp GlyCpg Ser Dtic Cpg B1 Antagonist (SEQ ID NO:16)    DOrnLys  Arg Pro ThzGly Cpg Ser Dtic Cpg B1 Antagonist (SEQ ID NO:17)    3PalLys  Arg ProHyp Gly Cpg Ser Dtic Cpg B1 Antagonist (SEQ ID NO:18)    4PalLys  ArgPro Hyp Gly Cpg Ser Dtic Cpg B1 Antagonist (SEQ ID NO:19)        Cha Arg Pro Hyp Gly Cpg Ser Dtic Cpg B1 Antagonist (SEQ ID NO:20)       2-NalArg Pro Hyp Gly Cpg Ser Dtic Cpg B1 Antagonist (SEQ IDNO:21)        Lys  Arg Pro Hyp Gly Cpg Ser Dtic Cpg B1 Antagonist (SEQID NO:22)    DLysLys  Arg Pro Hyp Gly Cpg Ser Dtic Cpg B1 Antagonist(SEQ ID NO:23)    Lys DOrn Arg Pro Hyp Gly Cpg Ser Dtic Cpg B1Antagonist (SEQ ID NO:24)    Lys Cha  Arg Pro Hyp Gly Cpg Ser Dtic CpgB1 Antagonist (SEQ ID NO:25)    Lys Abu  Arg Pro Hyp Gly Cpg Ser DticCpg B1 Antagonist (SEQ ID NO:26)    Lys 2-NalArg Pro Hyp Gly Cpg SerDtic Cpg    D- B1 Antagonist (SEQ ID NO:43)    Dab Lys  Arg Pro Hyp GlyCpg Ser Dtic Cpg    D- B1 Antagonist (SEQ ID NO:44) Ac Dab Lys  Arg ProHyp Gly Cpg Ser Dtic Cpg B1 Antagonist (SEQ ID NO:45)    DOrnLys  ArgPro Hyp Gly Cpg Ser Dtic Cpg B1 Antagonist (SEQ ID NO:46) Ac DOrnLys Arg Pro Hyp Gly Cpg Ser Dtic Cpg    D-    3′Pa B1 Antagonist (SEQ IDNO:47)    1   Lys  Arg Pro Hyp Gly Cpg Ser Dtic Cpg    D-    3′Pa B1Antagonist (SEQ ID NO:48) Ac 1   Lys  Arg Pro Hyp Gly Cpg Ser Dtic Cpg   D-  D-2- B1 Antagonist (SEQ ID NO:49)    Lys Nal  Arg Pro Hyp Gly CpgSer Dtic Cpg        D-2- B1 Antagonist (SEQ ID NO:50)    Lys Nal  ArgPro Hyp Gly Cpg Ser Dtic Cpg                             Me- B1Antagonist (SEQ ID NO:51)        DOrn Arg Oic Pro Gly Phe Ser D-β-NaIIle                             Me- B1 Antagonist (SEQ ID NO:52) Ac    DOrn Arg Oic Pro Gly Phe Ser D-β-NaI Ile                            Me- B1 Antagonist (SEQ ID NO:53)    DOrn LysArg Oic Pro Gly Phe Ser D-β-NaI Ile                             Me-B1 Antagonist (SEQ ID NO:54) Ac DOrnLys  Arg Oic Pro Gly Phe Ser D-β-NaIIle B1 Antagonist (SEQ ID NO:55)        Lys  Arg Pro Pro Gly Phe SerD-β-NaI Ile B1 Antagonist (SEQ ID NO:56) Ac     Lys  Arg Pro Pro Gly PheSer D-β-NaI Ile                             Me- B1 Antagonist (SEQ IDNO:57)        Orn  Arg Oic Pro Gly Phe Ser D-β-NaI Ile                            Me- B1 Antagonist (SEQ ID NO:58) Ac     Orn Arg Oic Pro Gly Phe Ser D-β-NaI Ile                             Me- B1Antagonist (SEQ ID NO:59)        Lys  Arg Oic Pro Gly Phe Ser D-β-NaIIle                             Me- B1 Antagonist (SEQ ID NO:60) Ac    Lys  Arg Oic Pro Gly Phe Ser D-β-NaI Ile

Vehicles. The term “vehicle” as used herein refers to a molecule thatprevents degradation and/or increases half-life, reduces toxicity,reduces immunogenicity, or increases biological activity of atherapeutic peptide or protein. Vehicles useful in the context of thepresent invention are known in the art (for example, see PCT PublicationWO 98/07746, which is hereby incorporated by reference in theirentirety) and are all readily available to those skilled in the art. Inthe context of the present invention, preferred vehicles include, butare not limited to, polymethylethylene-glycol,polyhydroxypropyleneglycol, polypropyleneglycols and oxides,polymethylpropyleneglycol, polyhydroxy-propyleneoxide, straight-chainand branched-chain polypropyleneglycols and derivatives thereof,polyethyleneglycol and polypropyleneglycol and the monomethyl ethers,monocetyl ethers, mono-n-butyl ethers, mono-t-butylethers and monooleylethers thereof, esters of polyalkyleneglycols with carboxylic acids anddehydration condensation products of the polyalkyleneglycols with aminesand other polyalkylene oxides and glycols and derivatives thereof,poly(vinylpyrrolidone), polyvinyl alcohol, poly(vinyl acetate), thecopolymer poly(vinyl acetate-co-vinyl alcohol), polyvinyloxazolidone,poly(vinylmethyloxazolidone and poly(vinyl methyl ether), poly(acrylicacid)s, poly(methacrylic acid)s, polyhydroxyethylmethacrylates,Poly(acrylamide and poly(methacrylamide) and other amides—thereof,poly(N,N-dimethylacrylamide), poly(N-isopropylacrylamide),poly(N-acetamidoacrylamide) and poly(N-acetamidomethacrylamide, andother N-substituted derivatives of the amides.

One aspect of the invention requires the presence of at least onevehicle (F) attached to a non-peptidyl linker moiety or an amino acidresidue of a peptidyl linker that is covalently fused to a peptide B1antagonist. In the context of the present invention, a preferred vehicleconstitutes a PEG molecule, as defined herein. An even more preferredvehicle constitutes a multivalent PEG molecule, as defined herein.

The vehicle- or PEG-conjugated molecules specifically disclosed orreferenced herein may be slightly modified within the regions denoted by(X¹)-(Y¹)_(n) (as defined supra.) to form an analog in accordance withthe invention, provided that antagonism of B1 is substantiallymaintained.

As between the vehicle- or PEG-conjugated peptides of the presentinvention and analogs thereof, it is preferable that no more than threenon-terminal residues in the (P) region are different. More preferably,analogs contemplated by the present invention include molecules with upto two amino acid substitions, insertions, or deletions at anyparticular non-terminal locus of the (P) region of the vehicle- orPEG-conjugated peptide of the present invention. Most preferably, thedivergence in sequence between a vehicle- or PEG-conjugated peptide ofthe present invention and a contemplated analog thereof, particularly inthe specified (P) region, is in the form of one or more “conservativemodifications”.

Linkers. The term “linker” as used herein refers to L¹ and, if present,L² as shown in either formula IV and V (supra.) and is abbreviatedherein by (L). Preferably, (L) is peptidyl in nature (i.e., made up ofamino acids linked together by peptide bonds) and made up of from 1 to 9amino acids. More preferably, (L) is made up of from 1 to 9 amino acids,wherein the amino acids are selected from the twenty naturally occurringamino acids. In an even more preferred embodiment the 1 to 9 amino acidsof the peptidyl linker are selected from cysteine, glycine, alanine,proline, arginine, asparagine, glutamine, and lysine. Even morepreferably, a peptidyl linker is made up of a majority of amino acidsthat are sterically unhindered, such as glycine and alanine linked by apeptide bond. Thus, preferred peptidyl linkers are poly(Gly)₁₋₈,particularly (Gly)₃ (SEQ ID NO:100), (Gly)₅ (SEQ ID NO:101) and (Gly)₇(SEQ ID NO:102), as well as poly(Gly-Ala)₂₋₄ and poly(Ala)₁₋₈. Otherspecific examples of peptidyl linkers include (Gly)₅Lys (SEQ ID NO:103),and (Gly)₅LysArg (SEQ ID NO:104). Other combinations of Gly and Ala arealso preferred. To explain the above nomenclature, for example,(Gly)₅Lys means Gly-Gly-Gly-Gly-Gly-Lys. A peptidyl linker may contain aN-terminal cysteine, another thiol, or nucleophile for conjugation witha vehicle. A more preferred linker contains an N-terminal cysteine orhomocysteine residue, or other 2-amino-ethanethiol or3-amino-propanethiol moiety for conjugation to maleimide, iodoacetaamideor thioester, functionalized vehicles. Treatment of the initial3-sulfanyl succinimide adduct (1c) formed by the reaction of a peptidewith maleimide activated PEG, with excess base converts the less stablesuccinimide adduct to the hydrolytically stable6-methylcarbamoyl-5-oxo-thiomorpholine-3-carboxamide form (1d, Scheme1). Alternatively, commercially available thioester or iodoacetamidoPEGs(Nektar Therapeutics, Huntsville, Ala.) may be used forchemoselective conjugation as depicted in Schemes 2 and 3.

Another preferred linker is a large, flexible linker comprising a randomGly/Ser/Thr sequence (GSGSATGGSGSTASSGSGSATH; SEQ ID NO:105) that isestimated to be about the size of a 1k PEG molecule. Additionally, apeptidyl linker may comprise a non-peptidyl segment such as a 6 carbonaliphatic molecule of the formula —CH₂—CH₂—CH2-CH2-CH2-CH2-(Rigidlinker: -AEAAAKEAAAKEAAAKAGG-).

Alternatively, a non-peptidyl linker containing a reactive nucleophilemay be present in X¹ and, if present, Y¹. For example, alkyl linkerssuch as —NH—(CH₂)_(s)—C(O)—, wherein s=2-20 could be used. These alkyllinkers may further be substituted by any non-sterically hindering 15group such as lower alkyl (e.g., C₁-C₆) lower acyl, halogen (e.g., Cl,Br), CN, NH₂, phenyl, etc. Exemplary non-peptidyl linkers are the PEGlinkers (shown below):

wherein n is such that the linker has a molecular weight of 100 to 5000kilodaltons (kD), preferably 100 to 500 kD m is =1-3. Preferably, anon-peptidyl linker is aromatic. The linkers may be altered to formderivatives in the same manner as described herein.

In addition, PEG moieties may be attached to the N-terminal amine orselected side chain amines by either reductive alkylation using PEGaldehydes or acylation using hydroxysuccinimido or carbonate esters ofPEG. Any of the linkers described above may be used in this approach.Alternatively, a suitably functionalized PEG may be attached directly toany of the peptide antagonists of the bradykinin B1 receptor as shown asSEQ ID NOS:5-26 or SEQ ID NOS: 43-60 or directly to an amino acidresidue of a peptidyl linker that is covalently fused to any of thepeptide antagonists of the bradykinin B1 receptor as shown as SEQ IDNOS:5-26 or 43-60.

It will be appreciated that, since the vehicle and/or the targetpeptides may be multivalent, it is possible by the process of theinvention to produce a variety of vehicle:peptide structures. By way ofexample, a univalent vehicle and a univalent peptide will produce a 1:1conjugate; a bivalent peptide and a univalent vehicle may formconjugates wherein the peptide conjugates bear two vehicle moietieswhereas a bivalent vehicle and a univalent peptide may produce specieswhere two peptide entities are linked to a single vehicle moiety; use ofhigher-valent vehicles can lead to the formation of clusters of peptideentities bound to a single vehicle moiety whereas higher-valent peptidesmay become encrusted with a plurality of vehicle moieties. The peptidemoieties may have more than one reactive group which will react with theactivated vehicle and the possibility of forming complex structures mustalways be considered; when it is desired to form simple structures suchas 1:1 adducts of vehicle and peptide, or to use bivalent vehicles toform peptide:vehicle:peptide adducts, it will be beneficial to usepredetermined ratios of activated vehicle and peptide material,predetermined concentrations thereof and to conduct the reaction underpredetermined conditions (such as duration, temperature, pH etc.) so asto form a proportion of the described product and then to separate thedescribed product from the other reaction products. The reactionconditions, proportions and concentrations of the reagents can beobtained by relatively simple trial-and-error experiments which arewithin the ability of an ordinarily skilled artisan with appropriatescaling-up as necessary. Purification and separation of the products issimilarly achieved by conventional techniques well known to thoseskilled in the art. However, as used in the specification and theappended claims, the singular forms “a”, “an”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a vehicle-conjugated peptide antagonist” or “aPEG-conjugated peptide antagonist” includes mixtures of such conjugatesand reference to “the method of treatment” includes reference to one ormore methods of treatment of the type which will be known to thoseskilled in the art or will become known to them upon reading thisspecification, and so forth.

Conventional PEGylations through the conjugation of mPEG-maleimide withthiol group of peptides and polypeptides having cysteine amino acidresidues are run in phosphate buffer with or without organic solvent.The high solubility of PEGylated peptides and polypeptides and potentialinstability of the pyrrolidin-2,5-dione ring in water have hampered theapplication of this method to large scale production and purification ofPEGylated peptides and proteins. Therefore, we disclose here a novelnon-aqueous conditions for the conjugation of mPEG-maleimide with thiolgroup of peptides and polypeptides having cysteine residues. The novelprocess results in moderate to high yields of PEGylated peptides andpolypeptides and combines the Michael addition and aminolysis intoone-pot (Scheme 4). Both conditions result in direct isolation ofPEGylated peptides and polypeptides through a precipitation.

In another embodiment of the process depicted in Scheme 4, inconjunction with any of the above or below embodiments, methanol (MeOH)may be substituted with a solvent comprising one or more of thefollowing solvents: methanol, ethanol, isopropyl alcohol, n-propanol,n-butanol, dichloromethane (DCM), acetonitrile (AcN), tetrahydrofuran(THF), dimethylformamide (DMF), dimethylacetamide (DMAc), andN-methylpyrrolidone (NMP).

In another embodiment of the process depicted in Scheme 4, inconjunction with any of the above or below embodiments, TBME may besubstituted with a solvent comprising one or more of the followingsolvents: diethyl ether, methyl isopropylether, and diisopropyl ether.

In another embodiment of the process depicted in Scheme 4, inconjunction with any of the above or below embodiments, reaction 1) asdepicted in Scheme 4 may be conducted at a temperature of about 20° C.to about 60° C. Preferably, reaction 1) as depicted in Scheme 4 can beconducted at a temperature of about 30° C. to about 50° C. Morepreferably, reaction 1) as depicted in Scheme 4 can be conducted at atemperature of about 35° C to about 45° C. Most preferably, reaction 1)as depicted in Scheme 4 can be conducted at room temperature.

In another embodiment of the process depicted in Scheme 4, inconjunction with any of the above or below embodiments, reaction 2) asdepicted in Scheme 4 may be conducted at a temperature of about 20° C.to about 60° C. Preferably, reaction 2) as depicted in Scheme 4 can beconducted at a temperature of about 30° C. to about 50° C. Morepreferably, reaction 1) as depicted in Scheme 4 can be conducted at atemperature of about 35° C. to about 45° C. Most preferably, reaction 2)as depicted in Scheme 4 can be conducted at room temperature.

In another embodiment of the process depicted in Scheme 4, inconjunction with any of the above or below embodiments, theprecipitation reaction as depicted in Scheme 4 may be conducted for atleast 10 minutes. More preferably, the precipitation reaction asdepicted in Scheme 4 may be conducted for at least 60 minutes. Mostpreferably, the precipitation reaction as depicted in Scheme 4 isconducted for about 60 minutes.

In another embodiment of the process depicted in Scheme 4, inconjunction with any of the above or below embodiments, theprecipitation, filtration, and/or purification reaction may be conductedmore than once.

In another embodiment of the process depicted in Scheme 4, inconjunction with any of the above or below embodiments, theprecipitation and/or purification reactions may be conducted more thanonce.

Partially protected peptides are especially useful reagents for thisstrategy as they enable selective modification of specific sites ofpolyfunctional peptides. The protecting groups are removed from the PEGconjugates using established deprotection methods well known by thoseskilled in the art of peptide synthesis. Partially protected peptidessuitable for this application may be prepared using orthogonalprotecting strategies well known to persons skilled in the art ofpeptide synthesis. An illustration of the synthesis of and conjugationof a partially protected peptide antagonist of the bradykinin B1receptor is depicted in Scheme 5. Analogs in which side chain aminesserve as sites of conjugation may be prepared from readily availableorthagonally protected basic amino acids.

Partially protected forms of B1 antagonist peptides such as those listedin Table 3 (SEQ ID NOS:5-60) may be conjugated to PEG moieties usingsimilar methods.

In one embodiment of the process depicted in Scheme 5, amine side chainsof the partially protected peptides 5c are masked by tert-butylcarbamoyl(Boc) moieties and the resulting peptides are reacted with any of thepreviously described PEG aldehydes in an organics solvents such as1,2-dichloroethane(DCE), N,N-dimethyl formamide(DMF) or mixturesthereof. The formation of the intermediate imine may be accelerated bythe addition of a dehydrating agent such as powdered 4° A molecularsieves. After stirring at room temperature for 1-24 hours, the resultingimine is reduced by the addition of 1-4 equivalents of sodium triacetoxyborohydride or sodium cyanoborohydride.

In another embodiment of the process depicted in Scheme 5, inconjunction with any of the above or below embodiments, reactions withpartially protected peptides such as 5c may be conducted at atemperature of about 20° C. to about 60° C. Preferably, reactions withpartially protected peptides such as 5c as depicted in Scheme 5 can beconducted at a temperature of about 20° C. to about 50° C. Morepreferably, reactions with partially protected peptides such as 5c asdepicted in Scheme 5 can be conducted at a temperature of about 35° C.to about 45° C. Most reactions with partially protected peptides such as5c as depicted in Scheme 5 can be conducted at room temperature.

In another embodiment of the process depicted in Scheme 5, amine sidechains of the partially protected peptides 5c are masked bytert-butylcarbamoyl (Boc) moieties and the resulting peptides arereacted with any of the previously described PEG N-hydroxysuccinimide orp-nitrophenyl ester PEG reagents in an organics solvents such as1,2-dichloroethane(DCE), N,N-dimethyl formamide(DMF), dichloromethane,N-methylpyrolidine(NMP) or mixtures thereof. Activated PEG esters may beeither monofunctional or linear bifunctional varieties both of which arecommericicially available from suppliers such as Nektar or NOF. Inaddition, branched polyfunctional PEG activated ester, containing 3-6hydroxysuccinimide or p-nitrophenylester moieties are especially usefulin preparing conjugates of the present invention.

In another embodiment of the process depicted in Scheme 5, inconjunction with any of the above or below embodiments, reactions withpartially protected peptides such as 5c may be conducted at atemperature of about 20° C. to about 60° C. with reaction times rangingfrom about 4 hours to about 10 days. Preferably, reactions withpartially protected peptides such as 5c as depicted in Scheme 5 can beconducted at a temperature of about 20° C. to about 50° C. with reactiontimes ranging from about 12 hours to about 5 days. More preferably,reactions with partially protected peptides such as 5c as depicted inScheme 5 can be conducted at a temperature of about 35° C. to about 45°C. with reaction times ranging from about 1 to about 5 days. Mostreactions with partially protected peptides such as 5c as depicted inScheme 5 can be conducted at room temperature with reaction timesranging from about 1 to about 4 days.

In another embodiment of the process depicted in Scheme 5, inconjunction with any of the above or below embodiments, the removal ofthe side chain protecting groups as depicted in Scheme 5 may beconducted at a temperature of about −20° C. to about 60° C. Thisreaction may be performed in a compatible solvent such asdichloromethane using between about 5% trifluoroacetic acid (TFA) and50% TFA by volume. Preferably, the acid mediated protecting groupremoval as depicted in Scheme 5 can be conducted at a temperature ofabout 0° C. to about 40° C., using about 10 to about 25% TFA by volume.More preferably, the acid mediated protecting group removal as depictedin Scheme 5 can be conducted at a temperature of about 0° C. to about25° C. using about 10% to about 20 % TFA by volume in dichloromethane.Most preferably, the acid mediated protecting group removal as depictedin Scheme 5 can be conducted at room temperature using about 20% byvolume in dichloromethane.

In another embodiment of the process depicted in Scheme 5, inconjunction with any of the above or below embodiments, the products 5ddepicted in Scheme 4 may be purified reverse phase HPLC, size exclusionchromatography, ion exchange chromatography or membrane dialysis. Morepreferably, a combination of two or more of the above mentionedpurification techniques may be used either in combination orsequentially to afford purified conjugates of the present invention.

In another embodiment of the process depicted in Scheme 5, inconjunction with any of the above or below embodiments, the purificationmethod may be conducted more than once.

Derivatives. Also contemplated herein are derivatives of the peptidesand/or conjugated peptides of the present invention. Such derivativesmay further improve the solubility, absorption, biological half-life,and the like, of the vehicle- or PEG-conjugated peptides disclosedherein. The added moieties may alternatively eliminate or attenuate anyundesirable characteristic of the peptides and/or conjugated peptidesdisclosed herein. Exemplary derivatives include vehicle- orPEG-conjugated peptides in which:

-   -   1. The peptides and/or conjugated peptide or some portion        thereof is cyclic. For example, the peptide portion of a peptide        and/or conjugated peptide may be modified to contain two or more        cysteine residues (e.g., in the peptidyl linker), which could        cyclize by disulfide bond formation. For citations to references        on the preparation of cyclized derivatives, see WO 00/24782.    -   2. The peptide and/or vehicle- or PEG-conjugated peptide is        cross-linked or is rendered capable of cross-linking between        molecules. For example, the peptide portion of a conjugated        peptide may be modified to contain one Cys residue and thereby        is able to form an intermolecular disulfide bond with a like        molecule.    -   3. One or more peptidyl [—C(O)NR—] linkages (peptide bonds) is        replaced by a non-peptidyl linkage. Exemplary non-peptidyl        linkages are —CH₂-carbamate [—CH₂—OC(O)NR—], phosphonate,        —CH₂-sulfonamide [—CH₂—S(O)₂NR—], urea [—NHC(O)NH—],        —CH₂-secondary amine, and alkylated peptide [—C(O)NR⁶— wherein        R⁶ is lower alkyl].    -   4. The N-terminal cysteine residue of a a conjugated peptide in        X¹ may be substituted with a N-terminal derivative group.        Exemplary N-terminal derivative groups include —NHR¹ where R¹ is        monoalkyl.

Derivatization with bifunctional agents is useful for cross-linking thevehicle-conjugated peptides or their functional derivatives to awater-insoluble support matrix or to other macromolecular vehicles.Commonly used cross-linking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]-propioimidate yield photo-activatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Carbohydrate (oligosaccharide) groups may conveniently be attached tosites that are known to be glycosylation sites in proteins. Generally,O-linked oligosaccharides are attached to serine (Ser) or threonine(Thr) residues, while N-linked oligo-saccharides are attached toasparagine (Asn) residues when they are part of the sequenceAsn-Aaa-Ser/Thr, where Aaa can be any amino acid except proline. Aaa ispreferably one of the nineteen naturally occurring amino acids otherthan proline. The structures of N-linked and O-linked oligosaccharidesand the sugar residues found in each type are different. One type ofsugar that is commonly found on both is N-acetylneuraminic acid(referred to as sialic acid). Sialic acid is usually the terminalresidue of both N-linked and O-linked oligosaccharides and, by virtue ofits negative charge, may confer acidic properties to the glycosylatedconjugated peptide. Such site(s) may be incorporated in the linker ofthe vehicle-conjugated peptides of the invention. Such sites may furtherbe glycosylated by synthetic or semi-synthetic procedures known in theart.

Other possible modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, oxidation of the sulfur atom in cysteine, methylation of thealpha-amino groups of lysine, arginine, and/or histidine side chains(Creighton, Proteins: Structure and Molecule Properties, W. H. Freeman &Co., San Francisco, pages 79-86. (1983)).

Unless otherwise disclosed herein, the synthesis of peptides and/orconjugated peptides described herein, including preparation ofappropriate amino acid derivatives, their activation and coupling toform peptides and methods for purification of peptides and determinationof their purity are included in the general body of knowledge of peptidechemistry, as generally described in Houben-Weyl “Methoden derOrganischen Chemie” Vol. 16, parts I & II, (1974) for solution phasesynthesis. For synthesis by the solid phase method, suitable techniquesare also well known in the art, and include those described inMerrifield, Chem. Polypeptides, pages 335-361 (Katsoyannis and Panayotiseditors) (1973); Merrifield, J. Am. Chem. Soc., Volume 85, page 2149(1963); Davis et al., Biochem. Intl., Volume 10, pages 394-414 (1985);Stewart and Young, Solid Phase Peptide Synthesis (1969); U.S. Pat. No.3,941,763; Finn et al., The Proteins (3d edition), Volume 2, pages105-253 (19.76); and Erickson et al., The Proteins (Third Edition),Volume 2, pages 257-527 (1976). A chemist skilled in the art of peptidesynthesis would be able to synthesize the described peptides by standardsolution methods or by manual or automatic solid phase methods. Solidphase synthesis is the preferred technique for making individualpeptides because of its cost-effectiveness.

Pharmaceutical Compositions

In General. The present invention also provides methods of usingpharmaceutical compositions of the inventive peptides and/orvehicle-conjugated peptides, e.g., in the prevention or treatment ofinflammation and pain (including, but not limited to, inflammatory painand associated hyperalgesia and allodynia). The peptides and/orvehicle-conjugated peptides of the invention also have therapeutic valuefor the prevention or treatment of other painful conditions associatedwith or mediated by B1 activation, including, but not limited to,thalamic pain syndrome, diabetes, toxins and chemotherapy, septic shock,arthritis, mixed-vascular and non-vascular syndromes, generalinflammation, arthritis, rheumatic diseases, lupus, osteoarthritis,inflammatory bowel disorders, inflammatory eye disorders, inflammatoryor unstable bladder disorders, psoriasis, skin complaints withinflammatory components, sunburn, carditis, inflammatory bowel disease,dermatitis, myositis, neuritis, collagen vascular diseases, chronicinflammatory conditions, epithelial tissue damage or dysfunction, herpessimplex, diabetic neuropathy pain, post-herpetic neuralgia, causalgia,sympathetically maintained pain, deafferentation syndromes, tensionheadache, angina, migraine, surgical pain, disturbances of visceralmotility at respiratory, genitourinary, gastrointestinal or vascularregions, wounds, burns, allergic rhinitis, asthma, allergic skinreactions, pruritis, vitiligo, general gastrointestinal disorders,colitis, gastric ulceration, duodenal ulcers, or vasomotor or allergicrhinitis.

The invention also provides for the use of the peptides and/orvehicle-conjugated peptides of the present invention for the preventionor treatment of acute pain, dental pain, back pain, lower back pain,pain from trauma, surgical pain, pain resulting from amputation orabscess, causalgia, demyelinating diseases, trigeminal neuralgia,cancer, chronic alcoholism, stroke, thalamic pain syndrome, diabetes,acquired immune deficiency syndrome (“AIDS”), toxins and chemotherapy,general headache, migraine, cluster headache, mixed-vascular andnon-vascular syndromes, tension headache, general inflammation,arthritis, rheumatic diseases, lupus, osteoarthritis, inflammatory boweldisorders, inflammatory eye disorders, inflammatory or unstable bladderdisorders, psoriasis, skin complaints with inflammatory components,sunburn, carditis, dermatitis, myositis, neuritis, collagen vasculardiseases, chronic inflammatory conditions, inflammatory pain andassociated hyperalgesia and allodynia, neuropathic pain and associatedhyperalgesia and allodynia, diabetic neuropathy pain, causalgia,sympathetically maintained pain, deafferentation syndromes, asthma,allergic rhinitis, epithelial tissue damage or dysfunction, herpessimplex, post-herpetic neuralgia, disturbances of visceral motility atrespiratory, genitourinary, gastrointestinal or vascular regions,wounds, burns, allergic skin reactions, pruritis, vitiligo, generalgastrointestinal disorders, colitis, gastric ulceration, duodenalulcers, and bronchial disorders.

Accordingly, the present invention also relates to the use of one ormore of the peptides and/or vehicle-conjugated peptides of the presentinvention in the manufacture of a medicament for the treatment of adisorder such as acute pain, dental pain, back pain, lower back pain,pain from trauma, surgical pain, pain resulting from amputation orabscess, causalgia, demyelinating diseases, trigeminal neuralgia,cancer, chronic alcoholism, stroke, thalamic pain syndrome, diabetes,acquired immune deficiency syndrome (“AIDS”), toxins and chemotherapy,general headache, migraine, cluster headache, mixed-vascular andnon-vascular syndromes, tension headache, general inflammation,arthritis, rheumatic diseases, lupus, osteoarthritis, inflammatory boweldisorders, inflammatory eye disorders, inflammatory or unstable bladderdisorders, psoriasis, skin complaints with inflammatory components,sunburn, carditis, dermatitis, myositis, neuritis, collagen vasculardiseases, chronic inflammatory conditions, inflammatory pain andassociated hyperalgesia and allodynia, neuropathic pain and associatedhyperalgesia and allodynia, diabetic neuropathy pain, causalgia,sympathetically maintained pain, deafferentation syndromes, asthma,allergic rhinitis, epithelial tissue damage or dysfunction, herpessimplex, post-herpetic neuralgia, disturbances of visceral motility atrespiratory, genitourinary, gastrointestinal or vascular regions,wounds, burns, allergic skin reactions, pruritis, vitiligo, generalgastrointestinal disorders, colitis, gastric ulceration, duodenalulcers, and bronchial disorders.

As used herein, “treatment” or “treating” is an approach for obtainingbeneficial or desired clinical results. For purposes of this invention,beneficial or desired clinical results include, but are not limited to,one or more of the following: improvement or alleviation of any aspectof pain and/or inflammation, including acute, chronic, inflammatory,neuropathic, or post-surgical pain. For purposes of this invention,beneficial or desired clinical results include, but are not limited to,one or more of the following: including lessening severity, alleviationof one or more symptoms associated with pain and/or inflammationincluding any aspect of pain and/or inflammation (such as shorteningduration of pain and/or inflammation, and/or reduction of painsensitivity or sensation).

Such pharmaceutical compositions or medicaments may be foradministration by injection, or for oral, pulmonary, nasal, transdermalor other forms of administration. In general, the invention encompassespharmaceutical compositions comprising effective amounts of at least onepeptide and/or at least one vehicle-conjugated peptide of the invention(in amounts effective to prevent, ameliorate, or abolish pain or any ofthe other medical conditions provided herein) together withpharmaceutically acceptable diluents, excipients, preservatives,solubilizers, emulsifiers, adjuvants and/or carriers. Such compositionsinclude diluents of various buffer content (e.g., Tris-HCl, acetate,phosphate), pH and ionic strength; additives such as detergents andsolubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants(e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g.,Thimerosol, benzyl alcohol) and bulking substances (e.g., lactose,mannitol); incorporation of the material into particulate preparationsof polymeric vehicle-conjugated peptides such as polylactic acid,polyglycolic acid, etc. or into liposomes. Hyaluronic acid may also beused, and this may have the effect of promoting sustained duration inthe circulation. Such compositions may further influence the physicalstate, stability, rate of in vivo release, and rate of in vivo clearanceof the vehicle-conjugated peptides of the present invention. See, forexample, Remington's Pharmaceutical Sciences, 18th Edition., MackPublishing Co., Easton, Pa., pages 1435-1712 (1990), which is hereinincorporated by reference. The compositions may be prepared in liquidform, or as a dried powder (such as lyophilized form). Implantablesustained release formulations are also contemplated, as are transdermalformulations.

Oral dosage forms. Contemplated for use herein are oral solid dosageforms, which are described generally in Chapter 89 of Remington'sPharmaceutical Sciences, above, which is herein incorporated byreference. Solid dosage forms include tablets, capsules, pills, trochesor lozenges, cachets or pellets. Also, liposomal or proteinoidencapsulation may be used to formulate the present compositions (suchas, for example, the proteinoid microspheres reported in U.S. Pat. No.4,925,673). Liposomal encapsulation may be used, and the liposomes maybe derivatized with various polymers (see, for example, U.S. Pat. No.5,013,556). A description of possible solid dosage forms is given inChapter 10 of Marshall, K., Modern Pharmaceutics, edited by G. S. Bankerand C. T. Rhodes (1979), herein incorporated by reference. In general,the formulation will include a vehicle-conjugated peptide of theinvention, as well as inert ingredients which allow for protectionagainst the stomach environment and release of the vehicle-conjugatedpeptide in the intestine.

Also specifically contemplated are oral dosage forms of the inventivepeptides and/or vehicle-conjugated peptides themselves. In this regard,if necessary, the peptides and/or vehicle-conjugated peptides may bechemically modified so that oral delivery is efficacious. It is alsopossible to use a salt of a modified aliphatic amino acid, such assodium N-(8-[2-hydroxybenzoyl]amino) caprylate (SNAC), as a carrier toenhance absorption of the vehicle-conjugated peptides of the invention.See U.S. Pat. No. 5,792,451, entitled “Oral Drug Delivery Compositionand Methods”.

The peptides and/or vehicle-conjugated peptides of the invention can beincluded in the formulation as fine multi-particulates in the form ofgranules or pellets of a particle size about one millimeter. Theformulation of the material for capsule administration could also be asa powder, as lightly compressed plugs, or even as tablets. Thetherapeutic could be prepared by compression.

Colorants and flavoring agents may all be included. For example, thepeptide and/or vehicle-conjugated peptide or any derivative thereof maybe formulated (such as by liposome or microsphere encapsulation) andthen further contained within an edible product, such as a refrigeratedbeverage containing colorants and flavoring agents.

One may dilute or increase the volume of the peptide and/orvehicle-conjugated peptide of the invention with an inert material.These diluents could include carbohydrates, especially, mannitol,α-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans andstarch. Certain inorganic salts may also be used as fillers, includingcalcium triphosphate, magnesium carbonate and sodium chloride. Somecommercially available diluents are Fast-Flo, Emdex, STA-Rx 1500,Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic intoa solid dosage form. Materials used as disintegrants include, but arenot limited to, starch, including the commercially availabledisintegrant based on starch, Explotab. Sodium starch glycolate,Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodiumalginate, gelatin, orange peel, acid carboxymethyl cellulose, naturalsponge and bentonite may also be used. Another form of the disintegrantsare the insoluble cationic exchange resins. Powdered gums may be used asdisintegrants and as binders, and these can include powdered gums suchas agar, Karaya or tragacanth. Alginic acid and its sodium salt are alsouseful as disintegrants.

Binders may be used to hold the components of the pharmaceuticalcomposition together to form a hard tablet, and they include materialsfrom natural products such as acacia, tragacanth, starch and gelatin.Others include methyl cellulose (MC), ethyl cellulose (EC) andcarboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) andhydroxypropylmethyl cellulose (HPMC) could both be used in alcoholicsolutions to granulate the therapeutic.

An anti-frictional agent may be included in the formulation to preventsticking during the formulating process. Lubricants may be used as alayer between the therapeutic and the die wall, and these can include,but are not limited to: stearic acid, including its magnesium andcalcium salts, polytetrafluoroethylene (PTFE), liquid paraffin,vegetable oils and waxes. Soluble lubricants may also be used such assodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol ofvarious molecular weights, Carbowax 4000 and 6000.

Glidants that might improve the flow properties of thevehicle-conjugated peptide during formulation and to aid rearrangementduring compression might be added. Such glidants may include starch,talc, pyrogenic silica and hydrated silicoaluminate.

To aid dissolution of the peptide and/or vehicle-conjugated peptide ofthe invention into the aqueous environment, a surfactant might be addedas a wetting agent. Such surfactants may include anionic detergents suchas sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctylsodium sulfonate. Cationic detergents may be used and can includebenzalkonium chloride or benzethonium chloride. The list of potentialnonionic detergents that may be included in the formulation assurfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylenehydrogenated castor oil 10, 50 and 60, glycerol monostearate,polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methylcellulose and carboxymethyl cellulose. These surfactants may be presentin the formulation either alone or as a mixture in different ratios.

Additives may also be included in the formulation to enhance uptake ofthe peptides and/or vehicle-conjugated peptide. Additives potentiallyhaving this property include various fatty acids, such as, for instance,oleic acid, linoleic acid and linolenic acid.

Controlled release formulation may be desirable. The peptide and/orvehicle-conjugated peptide of the invention may be incorporated into aninert matrix which permits release by either diffusion or leachingmechanisms, for example, gums. Slowly degenerating matrices may also beincorporated into the formulation, for example, alginates orpolysaccharides. Another form of a controlled release of the peptideand/or vehicle-conjugated peptide of the invention is by a method basedon the Oros therapeutic system (Alza Corp.), i.e., the drug is enclosedin a semipermeable membrane which allows water to enter and push drugout through a single small opening due to osmotic effects. Some entericcoatings also have a delayed release effect.

Pulmonary delivery forms. Also contemplated herein is pulmonary deliveryof a pharmaceutical composition in accordance with the invention. Thepeptide and/or vehicle-conjugated peptide (or derivatives thereof) isdelivered to the lungs of a mammal while inhaling and traverses acrossthe lung epithelial lining to the blood stream. Reports relating to thepulmonary delivery of macromolecules that may be helpful in this regardinclude Adjei et al., Pharma. Res., Volume 7, pages 565-569 (1990);Adjei et al., Internatl. J. Pharmaceutics, Volume 63, pages 135-144(1990) (leuprolide acetate); Braquet et al., J. Cardiovasc. Pharmacol.,Volume 13 (suppl. 5), s. 143-146 (1989) (endothelin-1); Hubbard et al.,Annals Int. Med., Volume 3, pages 206-12 (1989) (α1-antitrypsin); Smithet al., J. Clin. Invest., Volume 84, pages 1145-1146 (1989)(α1-proteinase); Oswein et al., “Aerosolization of Proteins”, Proc.Symp. Resp. Drug Delivery II, Keystone, Colo. (1990) (recombinant humangrowth hormone); Debs et al., J. Immunol., Volume 140, pages 3482-3488(1988) (interferon-γ and tumor necrosis factor α); and U.S. Pat. No.5,284,656 (granulocyte colony stimulating factor).

Contemplated for use in the practice of the invention are a wide rangeof mechanical devices designed for the pulmonary delivery of therapeuticproducts, including but not limited to nebulizers, metered doseinhalers, and powder inhalers, all of which are familiar to thoseskilled in the art. Some specific examples of commercially availabledevices suitable for the practice of the invention are the Ultraventnebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the AcornII nebulizer, manufactured by Marquest Medical Products, Englewood,Colo.; the Ventolin metered dose inhaler, manufactured by Glaxo Inc.,Research Triangle Park, N.C.; and the Spinhaler powder inhaler,manufactured by Fisons Corp., Bedford, Mass.

All such devices require the use of formulations suitable for thedispensing of the herein described peptides and/or vehicle-conjugatedpeptides and/or deerivatives thereof. Typically, each formulation isspecific to the type of device employed and may involve the use of anappropriate propellant material, in addition to diluents, excipientsadjuvants and/or carriers useful in therapy.

Pharmaceutically acceptable carriers for these pulmonary compositionsinclude carbohydrates such as trehalose, mannitol, xylitol, sucrose,lactose, and sorbitol. Other ingredients for use in formulations mayinclude DPPC, DOPE, DSPC and DOPC. Natural or synthetic surfactants maybe used. PEG may be used (even apart from its use in derivatizing thepeptide). Dextrans, such as cyclodextran, bile salts, cellulose andcellulose derivatives may also be used. Amino acids may be used, such asin a buffer formulation.

In addition, the use of liposomes, microcapsules or microspheres,inclusion complexes, or other types of carriers is contemplated.

Formulations suitable for use with a nebulizer, either jet or ultrasonictype, will typically comprise the described peptide and/orvehicle-conjugated peptide dissolved in water at a concentration ofabout 0.1 to 25 milligrams (mg) of biologically active agent permilliliter (ml) of solution. The formulation may also include a bufferand a simple sugar (e.g., for peptide stabilization and regulation ofosmotic pressure). The nebulizer formulation may also contain asurfactant, to reduce or prevent surface induced aggregation of theprotein caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing the described peptide and/orvehicle-conjugated peptide suspended in a propellant with the aid of asurfactant. The propellant may be any conventional material employed forthis purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, ahydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane,dichlorodifluoromethane, dichlorotetrafluoroethanol, and1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactantsinclude sorbitan trioleate and soya lecithin. Oleic acid may also beuseful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing the described peptides and/orvehicle-conjugated peptides and may also include a bulking agent, suchas lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol inamounts which facilitate dispersal of the powder from the device, e.g.,50 to 90% by weight of the formulation.

Nasal delivery forms. Nasal delivery of the peptide and/orvehicle-conjugated peptides is also contemplated. Nasal delivery allowsthe passage of the peptide and/or vehicle-conjugated peptides of theinvention to the blood stream directly after administering thetherapeutic product to the nose, without the necessity for deposition ofthe product in the lung. Formulations for nasal delivery include thosewith dextran or cyclodextran. Delivery via transport across other mucousmembranes is also contemplated.

Pump delivery. In certain embodiments of the present invention,localized delivery of the peptides and/or conjugated peptides of thepresent invention is contemplated for treating or preventing B1 mediateddisorders. One method of localized delivery contemplated by theinvention is injection of the agent at a local site at which the agentacts.

Another method for localized delivery includes inserting a catheter todirect the drug(s) to the desired body site, and using a pump to impel adrug(s) through the catheter. Externally worn drug pumps used with aninternally implanted catheter are well known to those skilled in theart.

Yet another method for localized delivery includes implantable drugdelivery devices. Implantable pumps have been developed to address thedisadvantages of techniques that use external pump and catheter systemsare are well-known to those skilled in the art. Implantable drugdelivery pumps often include a reservoir for storing the drug, aninjection port to enable injection of fresh drug preparations as wellremoval of old drug at regular intervals from the reservoir, andoptionally a catheter for delivering the drug to the desired site.Preferred implantable devices include, but are not limited to, the Durosimplant (Alza Corporation, Mountain View, Calif.), the SynchroMed I orII Infusion System (Medtronic, Inc., Minneapolis) and the like.

Additionally (or alternatively), the present invention provides peptidesand/or conjugated peptides for use in any of the various slow orsustained release formulations or microparticle formulations previouslymentioned hereinabove and/or known to the skilled artisan.

Dosages. Effective dosages of the peptides and/or conjugated peptides ofthe invention to be administered may be determined through procedureswell known to those in the art which address such parameters asbiological half-life, bioavailability, and toxicity. In preferredembodiments, an effective dosage range is determined by one skilled inthe art using data from routine in vitro and in vivo studies well knownto those skilled in the art. For example, in vitro cell culture assays,such as the exemplary assays described in Example 6 below will providedata from which one skilled in the art may readily determine the meaninhibitory concentration (IC) or mean effective concentration (EC) ofthe peptide or the conjugated peptide necessary to block some amount ofB1 induced activity (e.g., 50%, IC₅₀; or 90%, IC₉₀). Appropriate dosescan then be selected by one skilled in the art using pharmacokineticdata from one or more routine animal models, such as the exemplarypharmacokinetic data described in Example 9, below, so that a minimumplasma concentration (C_(min)) of the peptide is obtained which is equalto or exceeds the determined IC value. The dosage regimen involved in amethod for treating the involved disease or disorder will be determinedby the attending physician, considering various factors which modify theaction of therapeutic agents, such as the age, condition, body weight,sex and diet of the patient, the severity of the condition beingtreated, time of administration, and other clinical factors. Generally,the daily regimen should be in the range of 1.0-10000 micrograms (μg) ofthe peptide and/or vehicle-conjugated peptide per kilogram (kg) of bodyweight, preferably 1.0-1000 μg per kilogram of body weight, and mostpreferably 1.0-150 μg per kilogram of body weight.

Combination Therapy. In another aspect, the present invention includes amethod for treating (or, in other embodiments, preventing) pain and/orinflammation, or any condition or disorder associated with B1activation, comprising administering an amount of peptide and/orconjugated peptide of the present invention and an amount of an NSAID.The term “NSAID” refers to a non-steroidal anti-inflammatory compound.The relative amounts and ratios of peptide antagonist and/or conjugatedpeptide antagonist and NSAID may vary. In some embodiments, enough ofthe peptide(s) and/or conjugated peptide(s) will be administered so asto allow reduction of the normal dose of NSAID required to effect thesame degree of pain or inflammation amelioration. In some embodiments,enough of a peptide(s) and/or conjugated peptide(s) of the presentinvention will be administered so as to allow reduction of the normaldose of NSAID required to effect the same degree of pain or inflammationamelioration by at least about 5%, at least about 10%, at least about20%, at least about 30%, at least about 40%, at least about 50%, atleast about 60%, at least about 70%, at least about 80%, or at leastabout 90%, or more. This reduction may be reflected in terms of amountadministered at a given administration and/or amount administered over agiven period of time (reduced frequency).

In another aspect, the invention provides methods for enhancing NSAIDpain or inflammation treatment comprising administering an effectiveamount of an NSAID in conjunction with an effective amount of at leastone peptide and/or at least one conjugated peptide of the presentinvention. As used herein, “administration in conjunction” is also meantto encompass any circumstance wherein an NSAID and a peptide and/orconjugated peptide of the present invention are administered in aneffective amount to an individual. “Administration in conjunction”, asused herein, comprises simultaneous administration and/or administrationat different times. Administration in conjunction also encompassesadministration as a co-formulation (i.e., the peptide and/or conjugatedpeptide of the present invention and NSAID are present (combined) in thesame composition) and/or administration as separate compositions. It isunderstood that the peptide(s) and/or conjugated peptide(s) of thepresent invention and at least one NSAID can be administered atdifferent dosing frequencies and/or intervals. For example, a conjugatedpeptide of the present invention may be administered weekly, while anNSAID can be administered more frequently. It is understood that thepeptide(s) and/or conjugated peptide(s) of the present invention and theNSAID can be administered using the same route of administration ordifferent routes of administration, and that different dosing regimensmay change over the course of administration(s). Administration may evenbe before the onset of pain or inflammation. Therefore, in anotheraspect, the invention provides methods for treating, reducing incidenceof, palliating and/or delaying the development or progression of painand/or inflammation in an individual, said methods comprisingadministering an effective amount of at least one peptide and/or atleast one conjugated peptide of the present invention in conjunctionwith an effective amount of at least one NSAID. Such methods includetreating or preventing any pain and/or inflammation of any etiology,including pain and/or inflammation where the use of an NSAID isgenerally prescribed. Such methods are also suitable for treating orpreventing any condition or disorder previously mentioned hereinabove orhereinbelow as being mediated by or associated with B1 activation. Insome embodiments, the pain and/or inflammation is post-surgical pain. Insome embodiments, the pain and/or inflammation is associated with burnsor wounds. In other embodiments, the pain and/or inflammation isassociated with rheumatoid arthritis. In other embodiments, the painand/or inflammation is associated with osteoarthritis. In otherembodiments, the pain and/or inflammation is associated withpost-herpetic neuralgia. In some embodiments, the NSAID is selected fromthe group consisting of aspirin, acetominophen, ibuprofen, indomethacin,naproxen, naprosyn, diclofenac, ketoprofen, tolmetin, slindac, mefenamicacid, meclofenamic acid, diflunisal, Rufenisal, piroxim, sudoxicam,isoxicam, celecoxib, rofecoxib, DUP-697, flosulide, meloxicam,6methoxy-2 naphthylacetic acid, MK-966, nabumetone, nimesulide, NS-398,SC-5766, SC58215, T-614, or combinations thereof.

EXAMPLES

The following Examples are presented for illustrative purposes only andare not intended, nor should they be construed, as limiting theinvention in any manner. Those skilled in the art will appreciate thatmodifications and variations of the compounds disclosed herein can bemade without violating the spirit or scope of the present invention.Compounds according to the invention can be synthesized according to oneor more of the following methods. It should be noted that the generalprocedures are shown as it relates to preparation of compounds havingunspecified stereochemistry. However, such procedures are generallyapplicable to those compounds of a specific stereochemistry, e.g., wherethe stereochemistry about a group is (S) or (R). In addition, thecompounds having one stereochemistry (e.g., (R)) can often be utilizedto produce those having opposite stereochemistry (i.e., (S)) usingwell-known methods, for example, by inversion.

Example 1 Synthesis and Purification of B1 Receptor Peptide Antagonistsand PEG-Conjugated B1 Receptor Peptide Antagonists

Various peptides of the invention were synthesized using synthesistechniques well-known in the art. A preferred method of synthesizingvarious peptides of the invention uses a FMOC strategy with carbodiimideactivation as described below.

Part 1: Dissolve Fmoc-amino Acid to Resin using Carbodiimide Chemistry.

Fmoc-amino acid (3-4 equivalent) was dissolved in dry DCM/NMP mixture(NMP or DMF was used to aid complete dissolution). A solution ofN-hydroxybenzotriazole (HOBt, same equivalent to amino acid) in NMP wasadded to the amino acid solution. A solution ofN,N′-dicyclohexyl-carbodiimide (DCC, same equivalent to amino acid) inDCM was added to the amino acid solution. The solution was mixed forapproximately 20 minutes. The activated acid solution was then added toresin (if needed, precipitates were removed prior to addition). Thereaction was agitated until the resin was negative by the ninhydrintest. Upon completion of the coupling, the resin was collected andwashed with DMF several times.

Part 2: Remove N-terminal Fmoc from Peptide-resin.

Fmoc-protected peptidyl resin was treated with piperidine/DMF (2/8) for3 minutes. The resin was drained and treatment was repeated for 15 min.The resin was washed with DMF and then DCM several times. The resin wasair-dried if the next step involved cleavage of the peptide from theresin as described in step 3.

Part 3: TFA Cleavage and Deprotection.

The dried resin from Part 2 was placed in a flask and 10-25 ml/g resinof cleavage cocktail (95% TFA, 2.5% water, 1.5% triisopropylsilane and1% ethanedithiol) was added. After stirring the reaction for 3-4 hours,the resin was removed by filtration under reduced pressure and washedtwice with TFA. The combined filtrates were concentrated to ˜20% byrotary evaporation under reduced pressure. The liquid was cooled to −50°C., and precipitated with 10-fold volume of cold dry ether. Theprecipitate was collected. The peptide was then dissolved in awater/acetonitrile mixture containing 0.5% TFA and lyophilize. The crudeproduct was then purified using C18 HPLC, on a gradient from 10%acetonitrile/0.1% TFA in water to 50% acetonitrile/0.1% TFA in water.For 1 g of crude product, a 250×50 mm C18 column was used at a flow rateof 90 mL/min on an Agilent prep HPLC with dual wavelength detection at215 and 254 nm. The injection was fractionated and each fractionanalyzed by mass spectrometry. Tubes were pooled based on mass spec,concentrated under reduced pressure to remove acetonitrile, andlyophilized to obtain the peptide B1 antagonists as white powders.Characterization was accomplished by HPLC-MS and Maldi-TOF massdetermination.

Various PEG-conjugated peptides of the invention were prepared asfollows.

Various active bradykinin B1 receptor peptide antagonists selected fromthe group consisting of SEQ ID NOS:5-60) were synthesized with differentpeptidyl linkers at the N-terminus, and each containing a penultimatecysteine using the aforementioned methods (e.g., SEQ ID NOS:27-41).These peptide analogs were derivatized with different sizes andconfigurations of poly(ethylene glycol) (PEG) through site-directedcoupling of the maleimide activated polymer to the N-terminal cysteinethiol of the peptide analogs using, for example, Method A or Method Bdescribed below. The resultant PEG-peptide conjugates were purified byion exchange chromatography, concentrated by lyophilization ordiafiltration and dialyzed into buffer prior to in vitro and in vivobioassay.

Method A:

PEG-conjugated peptides were prepared by reacting a cysteine containingpeptide with PEG-maleimide in 50 mM NaHPO₄, 5 mM EDTA, pH 6.5 at 2.5-5mg/ml peptide and a reaction stoichiometry of 1.2-fold molar excess ofmaleimide:thiol. The reaction was stirred at room temperature (20-25 °C.) for 1-1.5 hr. Once complete, the reaction was quenched with a10-fold molar excess of P-mercaptoethanol (β-ME):maleimide and allowedto stir an additional 30-60 minutes at room temperature.

Progress of the reaction was monitored using reverse-phase HPLC(RP-HPLC) by injection of 5 μl of the reaction to a 4.6×250 mm, 5 micronC4 column (Grace Vydac, Columbia, Md.; cat. no.: #214TP54). Theunreacted peptide and PEG-peptide conjugate are eluted with a linear5-90% acetonitrile gradient in 0.1% trifluoroacetic acid.Typically, >90% of the peptide analog is consumed in the reaction.

Linear maleimide activated PEG polymers (MW=5 kD or 20 kD, PD=1.01-1.02)were provided by Shearwater Corp. or NOF Corp, (Toyko, Japan).

Purification:

The PEG-conjugated peptides were purified by cation exchangechromatography using SP Sepharose HP columns (Amersham Biosciences)pre-equilibrated with 10 mM NaOAc, 20% EtOH, pH 4. Prior to loading, thereaction mixtures were diluted 10-fold with 20% EtOH and the pH adjustedto 3.5 with glacial acetic acid. The diluted reaction mixtures wereloaded to an appropriate sized column such that a peptide:resin rationof 2.5 mg/ml was not exceeded.

The column was then washed with 2 column volumes (CVs) of 10 mM NaOAc,20% EtOH, pH 4 and eluted with a linear 0-200 mM NaCl gradient in 10 mMNaOAc, 20% EtOH, pH 4 over 10-20 CV. The unmodified peptide andPEG-peptide conjugate were detected by monitoring absorbance at either254 nm or 220 nm. Under these conditions, the excess PEG and β-ME werewashed out in the unbound flow-through fraction, the conjugate eluted ina broad peak starting ˜50 mM NaCl and the free peptide was wellresolved, eluting at ˜200 mM NaCl.

The eluted peak fractions were evaluated by RP-HPLC and pooled based onhomogeneity and retention times consistent with PEG-peptide conjugate.The pooled >conjugate peak was concentrated by drying, thenreconstituted in water and dialyzed against buffer. Alternativelydiafiltration may be used to concentrate and buffer exchange theconjugate.

The final pools of PEG-peptide conjugates were analyzed by RP-HPLC andwere typically ˜98% conjugate. Conjugate composition and concentrationswere determined by a combination of amino acid analyses, peptidesequencing, and absorbance spectroscopy.

The solution stability of compounds represented by 1c in Scheme 1 wasmonitored at ambient temperature in pH=7.2 phosphate buffered saline(PBS) over time using the CEX method described above (FIG. 1(A)).Compound 1c was shown to rapidly convert to id as well as to twoproducts resulting from hydrolysis of the succinimide moiety (structuresdetermined by a combination of IR, MS/MS and NMR experiments).

Method B:

mPEG-maleimide (1.0 eq.) was dissolved at 30° C. in anhydrous MeOH in a3-necked round bottom flask equipped with a mechanical stirrer,temperature probe, and a N₂ inlet. Upon total dissolution ofmPEG-maleimide, a peptide containing a N-terminal cysteine residue (1.3eq.) was added into the clear solution and stirred at rt for 3 h.Reverse phase HPLC shows disappearance of mPEG-maleimide and new peakfor the initial 3-sulfanyl-succinimide adduct. Next, ten equivalents ofdiisopropyl-ethylamine (Sigma-Aldrich Corp., St. Louis, Mo.) was addedinto the solution and stirred at 25° C. for at least 24 hours. Thereaction was monitored by ion exchange chromatography using TOSOHAASSP-5PW (20 μm) as stationary phase. CEX analysis indicated over 98%conversion with less 1.5% of the 3-sulfanyl-succinimide adductremaining. Tertiary butyl methyl ether (TBME) was added (twice thevolume methanol used in the reaction) and the resulting cloudy solutionwas stirred at room temperature for 1 hour. The white precipitate wasfiltered off and dried under vacuum at room temperature overnight toafford the crude 6-methyl-carbamoyl-5-oxo-thiomorpholine-3-carboxamidelinked product (1d).

Purification:

The above crude product was purified by RP-HPLC using MeOH—H₂O—AcOHsystem (c18 YMC ODS NQ as stationary phase) to afford the6-methyl-carbamoyl-5-oxo-thiomorpholine-3-carboxamide linked productwith a purity >98% by analytical reverse phase chromatography. The purefractions were combined and concentrated to dryness under vacuum and theresulting white residue was dissolved the minimum amount of warm MeOH(˜30° C.) sufficient to give a clear solution then treated with TBME(twice the volume of MeOH used). The resulting cloudy solution wasstirred at room temperature for 1 hour and precipitate was filtered off,dried under vacuum at room temperature for at least 16 hours. Pureproduct (1d) is obtained as an off-white in 74% overall yield with >98%CEX and RPC purity. Conjugate composition and peptide content weredetermined by a combination of amino acid analyses, peptide sequencing,multinuclear NMR methods and absorbance spectroscopy. The solutionstability of compound 1d was monitored at ambient temperature in pH=7phosphate buffered saline (PBS) was monitored over time using the CEXmethod described above. This compound proved significantly more stabile,with no significant changes noted over six days.

Analytical reverse phase (RP) and cation exchange (CEX) chromatographywas performed on Agilent 1100 HPLC systems with diode array or variablewavelength detectosr and thermostatted autosampler. Standardchromatographic conditions are outlined below. 1. RP-HPLC MethodConditions Column: YMC ODS-AQ, 3 μm, 120 Å, 4.6 × 100 mm Column Temp.40° C. Mobile Phases: A) 0.1% TFA in water B) 0.1% TFA in MeOH Flowrate: 1.1 mL/min Gradient: Time % B  0  5 10 40 30 95 35 95 35.1  5 40 5 Detection: UV at 220 nm Injection volume: 20 μL or 50 μL depending onthe sample concentration Sample concentration: 2.5 to 10 mg/mL Samplediluent: Dulbecco's PBS and other buffers used in the stability studies2. General Analytical Cation Exchange Chromatographic Method Column:TOSOH, TSK-GEL, SP-5PW, 10 μm, 7.5 × 75 mm Column Temp. 25° C. MobilePhases: A) 20 mM NaH₂PO₄ in water/EtOH (8:2), pH′ 3.5 B) 20 mM NaH₂PO₄and 0.5 M NaCl in water/ EtOH (8:2), pH 3.5 Flow rate: 1.0 mL/minGradient: Time B %  0  0  3  0 25  45 40 100 45 100 45.1  0 50  0Detection: UV at 220 nm Injection volume: 10 to 50 μL depending on thesample concentration Sample concentration: 2.5 to 10 mg/mL Samplediluent: Dulbecco's PBS and other buffers used in the stability studies

Example 2 Synthesis and Purification of PEG-Conjugated B1 ReceptorPeptide Antagonists using PEG Thioesters

PEG-conjugated peptides were prepared by reacting a cysteine containingpeptide with PEG-maleimide in 50 mM NaHPO₄, 5 mM EDTA, pH 7 at 2.5-5mg/ml peptide and a reaction stoichiometry of 1.2-fold molar excess ofmaleimide:thiol. The reaction was stirred at room temperature (20-25°C.) for 18-26 hours. Once complete, the reaction is quenched with a10-fold molar excess of β-mercaptoethanol (β-ME):maleimide and allowedto stir an additional 30-60 minutes at room temperature. The reactionswere purified as described for Method A in Example 1 above.

Example 3 Synthesis and Purification of PEG-Conjugated B1 ReceptorPeptide Antagonists using PEG Thioesters or Iodoacetates

PEG-conjugated peptides were prepared by reacting peptide containing aN-terminal cysteine residue with PEG-OPTE (ortho-pyridyl thio ester) in50 mM NaHPO₄, 5 mM EDTA, pH 7 at 2.5-5 mg/ml peptide and a reactionstoichiometry of 1.2-fold molar excess of activated PEG:peptide. Thereaction was stirred at room temperature (20-25° C.) for 18-26 hr. Oncecomplete, the reaction is quenched with a 10-fold molar excess ofcysteine:excess PEG reagent and allowed to stir an additional 30-60minutes at room temperature. The reactions were purified as described inMethod A of Example 1 above.

Alternatively, PEG-iodoacetamide may be used as described above to formconjugates in which the PEG moiety is attached via a thioether linkage(Scheme 3). In this case 1.5 molar equivalents of the activated PEGreaction is used and the reaction time is increased to 24 hours thereaction is quenched w/10 molar equivalents of β-mercaptoethanolpurified as described in the examples above.

Example 4 Synthesis and Purification of PEG-Conjugated B1 ReceptorPeptide Antagonists using PEG propionaldehyde

B1 receptor peptide antagonists such as any one of SEQ ID NOS:5-60 and27-41 can be selectively N-terminally modified with PEG using the methoddescribed in U.S. Pat. No. 5,824,784 (which is hereby incorporated byreference in its entirety). For example, the peptide as shown in SEQ IDNO:6 (245 mg, 0.14 mmol) was dissolved in 10 mL of solution containing100 mM NaH₂PO₄ and 60 mM NaCNBH₃. The mixture was cooled to 4° C. withover stirring and treated with 2.35 g of 20K mPEG porpionaldehyde(Nektar Therapuetics, Huntsville, Ala.). The mixture was stirred for 3days, then purified by RP and CEX chromatography as described in MethodB of Example 1.

Alternatively PEGs containing amine reactive functionalities may bereacted with partially protected B1 peptide antagonists according to themethods illustrated in Scheme 5. Following the conjugation reaction, theside chain protecting groups are cleaved using methods well known bythose skilled in the art of solid and solution phase peptide synthesis,and the resulting PEG-peptide constructs purified as described above.Multifunctional PEG aldehydes (3-6 reactive groups) may also be reactedwith excess molar amounts of protected peptides to afford multivalentPEG constructs in which multiple peptides are attached in aregiochemically and stiocheometerically defined manner.

Example 5 Synthesis and Purification of PEG-Conjugated B1 ReceptorPeptide Antagonists using PEG N hydroxy-succinimides

B1 receptor peptide antagonists such as any one of SEQ ID NOS:5-60 maybe selectively pegylated on a specific N terminal or side chain nitrogenatom using partially protected B1 peptide antagonists according to themethods illustrated in Scheme 5. For example a solution of partiallydecapeptide (1.43 g, 1.025 mmol) in 2.5 ml of anhydrous DMF was combinedwith 3.5 g (0.18 mmol) of Sunbright PTE-200GS (20 kD 4-armsuccinimidylgluterate, NOF, Tokyo, Japan) and 1.0 mL of diisopropylethylamine in 25 mL of dichloromethane. The resulting colorless solution wasstirred at room temperature for 2 days then evaporated at reducedpressure. The resulting residue was dissolved in 25 mL of deionizedwater and placed in a 10,000 MW cutoff dialysis membrane (Pierce,Rockford Ill., USA). The compound was dialyzed against water for 24hours (3 buffer changes), then lyophilized to afford the protectedtetravalenent PEG product. The resulting white solid was dissolved in 60mL dichloromethane and treated with 20 mL of anhydrous TFA. Afterstirring at roomtemperature for 2 days the reaction mixture wasevaporated at reduced pressure then dissolved in dialyzed as above. Thedialyzed material was lyophilized then purified by ion exchangechromatography as previously described to afford the tetravalent productas a white solid. In a similar fashion PEGs containing 1-6succinimidylgluterate moieties may be used to prepare mono orpolyfunctional peptide constructs. TABLE 4a X¹ Peptides SEQ ID NO:Sequence of X¹ Peptide 27 {N} CGGGKRPPGFSPL {C} 28 {N} CGGGGGKRPPGFSPL{C} 29 {N} CGGGGGKKRPGFSPL {C} 30 {N} CGGGGGKRKRPPGFSPL {C} 31 {N}CG—CH2—CH2—CH2—CH2—CH2—CH2—KRPPGFSPL {C} 32 {N}CGGGGGKKRPPG[AMeF]S[D-β-Nal]I {C} 33{N}CGGGGGKKRP[Hyp]G[Cpg]S[DTic][Cpg]{C} 34{N}CGGGGGGGKKRP[Hyp]G[Cpg]S[DTic][CPG] {C} 35 {N}ac-CGGGGGKKRP[Hyp]G[Cpg]S[DTic][Cpg]{C} 36{N}KKRP[Hyp]G[Cpg]S[DTic][Cpg] {C} 37{N}acyl-KKRP[Hyp]G[Cpg]S[DTic][Cpg] {C} 38 {N} CKRPPGFSPL {C} 39{N}CGGGGG[DOrn]KRP[Hyp]G[Cpg]S[DTic][Cpg]{C} 40{N}CGGGGG[DOrn]KRP[Thz]G[Cpg]S[DTic][Cpg]{C} 41{N}CGGGGGK[DOrn]RP[Hyp]G[Cpg]S[DTic][Cpg]{C}

TABLE 4b Y¹ Peptides SEQ ID NO: Sequence of Y¹ Peptide 42 {N}GGGGGKKRPPGFSPL {C}

Example 6 In vitro B1-Inhibition Activity of Peptide PEG-conjugatedPeptide Antagonists of B1 Activity

Peptides and/or conjugated peptides capable of selectively inhibiting B1activity as compared to B2 activity were identified using assays such asthose described in Sections A, B, and C below.

A. In vitro Assay of Human B1 Receptor Function using Calcium Flux:

Activation of the G_(q) linked B1 receptor results in an increase inintracellular calcium. The calcium sensitive photoprotein aequorin can,therefore, be used as an indicator of B1 receptor activation. Aequorinis a 21-kDa photoprotein that forms a bioluminescent complex when linkedto the chromophore cofactor coelenterazine. Following the binding ofcalcium to this complex, an oxidation reaction of coelenterazine resultsin the production of apoaequorin, coelenteramide, CO₂, and light thatcan be detected by conventional luminometry

A stable CHO D-/human B1 receptor (GenBank Accession no.AJ238044)/Aequorin cell line was established and the cells weremaintained in suspension in spinner bottles containing a 1:1 ratio ofDMEM and HAM F12 (Gibco 11765-047), high glucose (Gibco 11965-084), 10%Heat Inactivated Dialyzed serum (Gibco 26300-061), 1X Non-EssentialAmino Acids (Gibco 11140-050), 1X Glutamine-Pen-Strep (Gibco 10378-016),and Hygromycin, 300 μg/ml (Roche 843555). Fifteen to twenty four hoursprior to the luminometer assay, 25,000 cells/well (2.5E6 cells/10ml/plate) are plated in 96-well black-sided clear bottom assay plates(Costar #3904).

Media is removed from the wells and replaced with 60 μl of serum freeHAM's F12 with 30 mM HEPES (pH 7.5) and 15 μM coelenterazine(Coelenterazine h Luciferin #90608; Assay Designs (Ann Arbor, Mich.).The plates are then incubated for 1.5-2 hours. Ten point IC₅₀ compoundplates containing 1:3 or 1:5 dilutions of antagonist compounds and anagonist activator plate (20 nM des-Arg10-Kallidin final concentration,EC₈₀) are prepared using Ham's F12 with 30 mM HEPES, pH 7.5. Followingcoelenterazine incubation, an automated flash-luminometer platform isused to dispense the B1 antagonist compounds to the cell plate, a CCDcamera situated underneath the cell plate takes 12 images of the cellplate at 5 second intervals to determine if there is any agonistactivity with the compounds. The hB1 agonist, des-Arg₁₀-Kallidin, isthen added to the cell plate and another 12 images are recorded todetermine the IC₅₀of the antagonist(s).

B. In vitro Assay of hB2 Receptor Function using Calcium Flux:

The intracellular calcium flux induced by hB2 receptor activation isanalyzed using a hB2 recombinant cell line (CHO—K1) purchased fromPerkinElmer (Wellesley, Mass.; catalog no.: RBHB2C000EA) on afluorometric imaging plate reader (FLIPR). The cells are cultured inT225 flask containing Ham's F12 Nutrient Mixture (Invitrogen Corp.,Carlsbad, Calif.; catalog no.: 11765-047), 10% Fetal Clone II BovineSerum (HyClone, Logan, Utah; catalog no.: SH3006603), 1 mM Sodiumpyruvate (100 mM stock, Invitrogen Corp., catalog no.: 12454-013), and0.4 mg/ml Geneticin (G418; 50 mg/ml active geneticin, Invitrogen,catalog no.: 10131-207). Culture medium is changed every other day. 24hrs prior to the FLIPR assay, the hB2/CHO cells are washed once with PBS(Invitrogen) and 10 ml of Versene (1:5000, Invitrogen, catalog no.:15040-066) is added to each flask. After a 5 minute incubation at 37°C., Versene is removed and cells are detached from the flask andresuspended in culture medium. Cells are counted and 25,000 cells/wellare plated in 96-well black-sided clear bottom assay plates (Costar,Acton, Mass.; catalog no.: 3904). Cells are incubated in a 37° C. CO₂incubator overnight.

The media is aspirated from the cells and replaced with 65 μl ofdye-loading buffer. The loading buffer is prepared by diluting a stocksolution of 0.5 mM Fluo-4 AM (Molecular Probes, Eugene, Oreg.) dissolvedin DMSO containing 10% [w/v] pluronic acid to a concentration of 1 μM inClear Dulbecco's Modified Eagle Medium (DMEM) containing 0.1% BSA, 20 mMHEPES, and 2.5 mM probenecid (probenecid inhibits activity of the aniontransport protein, and thus improves dye loading in the cells). Thecells are dye-loaded for 1 hour at room temperature. The excess dye isremoved by washing the cells two times with assay buffer. The assaybuffer consists of Hank's Balanced Salt Solution (HBSS) containing 20 mMHEPES, 0.1% BSA, and 2.5 mM probenecid. After the wash cycles, a volumeof 100 μL is left in each well, and the plate is ready to be assayed inthe FLIPR System. Single point (10 μM final concentration) POCantagonist compound plates or ten point IC₅₀ compound plates containing1:3 or 1:5 dilutions of antagonist compounds and an agonist activatorplate (0.3 nM bradykinin final concentration, EC₈₀) are prepared usingassay buffer. The cell plate and the compound plates are loaded onto theFLIPR and during the assay, fluorescence readings are takensimultaneously from all 96 wells of the cell plate. Ten 1-secondreadings are taken to establish a stable baseline for each well, then 25μL from the B1 antagonist plate is rapidly (50 μL/sec.) added. Thefluorescence signal is measured in 1-second (1 minute) followed by6-second (2 minutes) intervals for a total of 3 minutes to determine ifthere is any agonist activity with the compounds. The B2 agonist,bradykinin, is then added to the cell plate and another 3 minutes arerecorded to determine the percent inhibition at 10 μM (POC plates) orthe IC₅₀ of the antagonist.

The IC₅₀ values for vehicle- or PEG-conjugated peptides tested in thehB1 aequorin assay were on average the slightly reduced in vitroactivity conferred to peptides conjugated to larger PEG polymers. Forexample, the peptide represented by SEQ ID NO:36 and its acetylated formrepresented by SEQ ID NO:37, resulted in an IC₅₀ of 3.0 nM (±5 nM, n=8)and 3.2 nM (±3.2 nM, n=9), respectively at the hB1 receptor. However,the same peptide conjugated to PEG as described herein demonstratedapproximately a 10-fold increase in IC₅₀ The native, acetylated, andPEG-conjugate forms of the peptide were inactive up to 10 μM in the hB2FLIPR assay. None of the compounds showed agonist activity at either thehB1 or hB2 receptor.

C. Tissue Based in vitro Assays of hB1 Receptor Binding Peptides:

The antagonist activity and selectivity for bradykinin B1 receptor ofthe peptides and/or vehicle-conjugated peptides of the present inventionwere determined with the in vitro human umbilical Vein (HUV)contractility assay described below:

Endothelium-denuded vessels were suspended in 20-ml organ bathscontaining an oxygenated (95% O₂ and 5% CO₂) and pre-warmed (37° C.)standard physiological salt solution of the following composition (inmM): NaCl 118.0, KCl 4.7, MgSO₄ 1.2, CaCl₂ 2.5, KH₂PO₄ 1.2, NaHCO₃ 25.0and glucose 11.0 (pH 7.4). High K+ solutions (80 mM KCl) were preparedby equimolar replacement of NaCl with KCl. Hoe 140 (1 μM), mergetpa (1μM) and captopril (10 μM) were also present throughout the experimentsto block the B2 receptors and to prevent peptide degradation,respectively. The tissues were connected to force transducers forisometric tension recordings then allowed to equilibrate for asufficient time under an optimal resting tension. The experiments werecarried out using semi-automated isolated organ systems possessing eightorgan baths each, with multichannel data acquisition. The tissues wereexposed first to a high K+ solution (80 mM KCl) to obtain a controlcontraction. Following washings and a subsequent 60-min equilibrationperiod, the tissues were exposed to cumulative increasing concentrationsof the reference agonist Lys-desArg9-BK to obtain concentration-responsecurves in the absence (control preparations) or presence of variousconcentrations of the test compounds or the reference antagonistLys-desArg9[Leu8]-BK (test preparations), which were added min beforethe exposure to Lys-desArg9-BK. A concentration-response curve toLys-desArg9-BK was generated in each preparation.

The parameter measured was the maximal change in tension induced by eachagonist concentration and the results expressed as a percent of thecontrol responses to KCl. The EC₅₀ values of the agonist (concentrationproducing a half-maximum response) were calculated by linear regressionanalysis of its concentration-response curves. The antagonist potenciesof the test compounds and Lys-desArg9[Leu8]-BK were evaluated in termsof pA2 values (-log concentration producing a two-fold rightward shiftof the agonist concentration-response curve), which were calculatedaccording to Van Rossum (Van Rossum, J. M., Cumulative dose-responsecurves. II. Technique for the making of dose-response curves in isolatedorgans and the evaluation of drug parameters. Arch. Int. Pharmacodyn.Ther., 143:299-330 (1963)). The pA2 values were calculated using onlyantagonist concentrations that caused a significant rightward shift ofthe agonist concentration-response curve. The pA2 values are given asthe mean ±s.e.m. of three determinations. Statistical significance ofthe differences was determined using Student's t test and p values <0.05were considered statistically significant.

D. In vitro B1-Inhibition Activity of Peptides and/or ConjugatedPeptides

The effectiveness of the peptides and/or conjugated peptides asinhibitors of B1 activity (i.e., B1 “neutralization”) can also beevaluated by measuring the ability of each peptide and/or conjugatedpeptide to block B1 stimulated CGRP and substance P release and calciumsignaling in Dorsal Root Ganglion (DRG) neuronal cultures.

Dorsal Root Ganglion Neuronal Cultures. Dorsal root ganglia aredissected one by one under aseptic conditions from all spinal segmentsof embryonic 19-day old (E19) rats that are surgically removed from theuterus of timed-pregnant, terminally anesthetized Sprague-Dawley rats(Charles River, Wilmington, Mass.). DRG are collected in ice-cold L-15media (GibcoBRL, Grand Island, N.Y.) containing 5% heat inactivatedhorse serum (GibcoBRL), and any loose connective tissue and bloodvessels are removed. The DRG are rinsed twice in Ca²⁺— and Mg²⁺-freeDulbecco's phosphate buffered saline (DPBS), pH 7.4 (GibcoBRL). The DRGare then dissociated into single cell suspension using a papaindissociation system (Worthington Biochemical Corp., Freehold, N.J.).Briefly, DRG are incubated in a digestion solution containing 20 U/ml ofpapain in Earle's Balanced Salt Solution (EBSS) at 37° C. for fiftyminutes. Cells are dissociated by trituration through fire-polishedPasteur pipettes in a dissociation medium consisting of MEM/Ham's F12,1:1, 1 mg/ml ovomucoid inhibitor and 1 mg/ml ovalbumin, and 0.005%deoxyribonuclease I (DNase). The dissociated cells are pelleted at 200×gfor five minutes and re-suspended in EBSS containing 1 mg/ml ovomucoidinhibitor, 1 mg/ml ovalbumin and 0.005% DNase. Cell suspension iscentrifuged through a gradient solution containing 10 mg/ml ovomucoidinhibitor, 10 mg/ml ovalbumin at 200×g for six minutes to remove celldebris, and then filtered through a 88-μm nylon mesh (Fisher Scientific,Pittsburgh, Pa.) to remove any clumps. Cell number is determined with ahemocytometer, and cells are seeded into poly-ornithine 100 μg/ml(Sigma, St. Louis, Mo.) and mouse laminin 1 μg/ml (GibcoBRL)-coated96-well plates at 10×10³ cells/well in complete medium. The completemedium consists of minimal essential medium (MEM) and Ham's F12, 1:1,penicillin (100 U/ml), streptomycin (100 μg/ml), and 10% heatinactivated horse serum (GibcoBRL). The cultures are kept at 37° C., 5%CO₂ and 100% humidity. For controlling the growth of non-neuronal cells,5-fluoro-2′-deoxyuridine (75 μM) and uridine (180 μM) are included inthe medium.

Treatment with B1 and anti-B1 peptides and/or anti-B1 conjugatedpeptides. Two hours after plating, cells are treated with recombinanthuman β-B1 or recombinant rat β-B1 at a concentration of 10 ng/ml (0.38nM). Positive controls comprising serial-diluted anti-B1 antibody (R&DSystems, Minneapolis, Minn.) are applied to each culture plate. Testpeptides or test conjugated peptides (e.g., from Example 1) are added atten concentrations using 3.16-fold serial dilutions. All samples arediluted in complete medium before being added to the cultures.Incubation time is generally around 40 hours prior to measurement of VR1expression.

Measurement of VR1 Expression in DRG Neurons. Cultures are fixed with 4%paraformaldehyde in Hanks' balanced salt solution for fifteen minutes,blocked with Superblock (Pierce, Rockford, Ill.), and permeabilized with0.25% Nonidet P-40 (Sigma) in Tris.HCl (Sigma)-buffered saline (TBS) forone hour at room temperature. Cultures are rinsed once with TBScontaining 0.1% Tween 20 (Sigma) and incubated with rabbit anti-VR1 IgG(prepared at Amgen) for one and one-half hours at room temperature,followed by incubation of Eu-labeled anti-rabbit second antibody (WallacOy, Turku, Finland) for one hour at room temperature. Washes with TBS(3×five minutes with slow shaking) are applied after each antibodyincubation. Enhance solution (150 μl/well, Wallac Oy) is added to thecultures. The fluorescence signal is then measured in a time-resolvedfluorometer (Wallac Oy). VR1 expression in samples treated with thevehicle-conjugated peptides is determined by comparing to a standardcurve of B1 titration from 0-1000 ng/ml. Percent inhibition (compared tomaximum possible inhibition) of B1 effect on VR1 expression in DRGneurons is determined by comparing to controls that are not B1-treated.

Impaired receptor binding and functional activity for each of thePEG-conjugated peptide B1 antagonists was directly related to the sizeof the PEG group added and ranged from ˜5-200 fold reductions inpotency. Polyglycine linkers of ˜5-7 residues worked well to preservefunctional while longer linkers either showed little improvement(“flexible linker”) or proved to be a detriment to activity (“rigidlinker”). Finally, Table 8 illustrates the breadth of this applicationwith a variety of different PEG-conjugated peptide B1 antagonists. TABLE8 Binding affinity (Ki) and Functional potency (IC₅₀) at the human B1receptor (hB1) for peptide and PEGylated peptide antagonists hB1 hB1Activated PEG Peptides Peptide Ki IC₅₀ reagent per PEG (X¹) − (Y¹)0 or 1(nM) (nM) MeO-20K-Maleimide^(a) 1 SEQ ID NO: 28 114 110MeO-20K-Maleimide^(a) 1 SEQ ID NO: 29 252 237 MeO-20K-Maleimide^(a) 1SEQ ID NO: 30 230 61 MeO-20K-Maleimide^(a) 1 SEQ ID NOS: 29 + 22 54 42MeO-20K-Maleimide^(a) 1 SEQ ID NO: 32 9 69 MeO-20K-Maleimide^(a) 1 SEQID NO: 33 75 98 MeO-20K- 1 SEQ ID NO: 13 1 35 propionaldehyde^(b)Maleimide-20K- 2 SEQ ID NO: 33 11 77 Maleimide^(a) MeO-20K SPA^(c) 1 SEQID NO: 13 10 52 Tetrakis-20K-SPA^(c) 4 SEQ ID NO: 13 0.14 10Tetrakis-20K-SPA^(d) 4 SEQ ID NO: 13 0.14 10 none NA SEQ ID NO: 13 0.100.5 none NA SEQ ID NO: 15 0.19 1 none NA SEQ ID NO: 49 0.77 0.5 none NASEQ ID NO: 50 0.2 6 none NA SEQ ID NO: 22 0.38 2 None NA SEQ ID NO: 370.8 1^(a)Generated by one-pot process described in Scheme 4;^(b)Reductive amination at N-terminal residue, epsilon amine;^(c)acylated on the N-terminal residue, epsilon amine;^(d)acylated on the N-terminal residue, alpha amine

Example 7 Determination of Stability of Peptides and/or ConjugatedPeptides

A. Rat Kidney Brush Border Microvilli Assay

Kidney membranes are prepared according to the procedure set forth in byBooth et al. (Biochemical Journal, 142:575 (1974)). Proteinconcentrations are determined by the method of Bradford (Anal.Biochemistry., 20 72:248-254 (1976)).

B. Rat or Human Lung S9 Homogenate Assay Rat or Human lung is preparedas described by Skidgel et al. (Biochemical Pharmacology 33: 3471(1984)).

The test compounds are prepared at 1 mM concentration in a PBS solution(pH=7.1). Test compounds are added to a preparation of tissue fromprocedure A or B (final protein concentration of 2 mg/ml) and incubatedat 37° C. At various time points, the protein is precipitated withacetonitrile, 0.1M HCl in acetonitrile or 10% TFA in water. Theprecipitate is removed by centrifugation, and the filtrate furtherfiltered through a 0.1 μM membrane. The sample is then analyzed byreverse phase HPLC (4.6×300 mm Novapak HR C18 (Waters Corporation,Milford, Mass.) flow=1 mL/min, linear gradient from 10% ACN (0.1% Formicacid)—90% water (0.1% Formic acid) to 50% ACN (0.1% Formic acid)—50%water (0.1% Formic acid over 20 minutes) with mass spectroscopydetection. The concentration of the test compound at time T relative tothe internal standard is fitted to a first order loss function([compound]_(t)=[compound]₀ (1-e^((−kt))); “[compound]0” and“(compound]t” are the concentration of test compound at time zero andthe concentration of test compound at the time the sample is withdrawn,respectively; the variable “t” is the time the sample is withdrawn foranalysis; and k is the rate of test compound concentration change). Thevariable “k” is determined by using a non-linear regression approachsupplied by the JMP Statistical software package. Given that the testcompound concentration decreases over time, the values of “k” arenegative. The half-life is calculated from the model derived value of“k” using the following formula:T½(Ln 2)/k

Example 8 In vivo Antinociceptive Activity of anti-B1 Peptides andVehicle-Conjugated anti-B1 Peptides in Rat and Monkey Pain Models

A. Rat Neuropathic Pain Model. Male Sprague-Dawley rats (200 g) areanesthetized with isoflurane inhalant anesthesia and the left lumbarspinal nerves at the level of L5 and L6 are tightly ligated (4-0 silksuture) distal to the dorsal root ganglion and prior to entrance intothe sciatic nerve, as first described by Kim and Chung (An experimentalmodel for peripheral neuropathy produced by segmental spinal nerveligation in the rat. Pain 50:355-363, (1992)). The incisions are closedand the rats are allowed to recover. This procedure results inmechanical (tactile) allodynia in the left hind paw as assessed byrecording the pressure at which the affected paw (ipsilateral to thesite of nerve injury) was withdrawn from graded stimuli (von Freyfilaments ranging from 4.0 to 148.1 mN) applied perpendicularly to theplantar surface of the paw (between the footpads) through wire-meshobservation cages. A paw withdrawal threshold (PWT) was determined bysequentially increasing and decreasing the stimulus strength andanalyzing withdrawal data using a Dixon non-parametric test, asdescribed by Chaplan, S. R., et al. (Quantitative assessment of tactileallodynia in the rat paw. J. Neurosci. Meth, 53:55-63 (1994)).

Normal rats and sham surgery rats (nerves isolated but not ligated)withstand at least 148.1 mN (equivalent to 15 g) of pressure withoutresponding. Spinal nerve ligated rats respond to as little as 4.0 mN(equivalent to 0.41 g) of pressure on the affected paw. Rats areincluded in the study only if they did not exhibit motor dysfunction(e.g., paw dragging or dropping) and their PWT was below 39.2 mN(equivalent to 4.0 g). At least seven days after surgery rats aretreated with test peptides or test vehicle-conjugated peptides (usuallya screening dose of about 1 mg/kg and about 60 mg/kg, respectively) orcontrol diluent (PBS) once by s.c. injection and PWT was determined eachday thereafter for 7 days.

B. Rat CFA Inflammatory Pain Model. Male Sprague-Dawley rats (200 g) arelightly anesthetized with isoflurane inhalant anesthesia and the lefthindpaw is injected with complete Freund's adjuvant (CFA), 0.15 ml. Thisprocedure results in mechanical (tactile) allodynia in the left hind pawas assessed by recording the pressure at which the affected paw iswithdrawn from graded stimuli (von Frey filaments ranging from 4.0 to148.1 mN) applied perpendicularly to the plantar surface of the paw(between the footpads) through wire-mesh observation cages. PWT isdetermined by sequentially increasing and decreasing the stimulusstrength and analyzing withdrawal data using a Dixon non-parametrictest, as described by Chaplan et al. (1994). Rats are included in thestudy only if they do not exhibit motor dysfunction (e.g., paw draggingor dropping) or broken skin and their PWT is below 39.2 mN (equivalentto 4.0 g). At least seven days after CFA injection rats are treated withtest peptides and/or test vehicle-conjugated peptides (usually ascreening dose of 60 mg/kg) or control solution (PBS) once by s.c.injection and PWT is determined each day thereafter for 7 days. Averagepaw withdrawal threshold (PWT) was converted to percent of maximumpossible effect (% MPE) using the following formula: % MPE=100*(PWT oftreated rats−PWT of control rats)/(15-PWT of control rats). Thus, thecutoff value of 15 g (148.1 mN) is equivalent to 100% of the MPE and thecontrol response is equivalent to 0% MPE.

Preferred peptides and vehicle-conjugated peptides of the presentinvention are expected to produce an antinociceptive effect with a PDrelationship at a screening dose of about 1 mg/kg and about 60 mg/kg,respectively.

B. Green Monkey LPS Inflammation Model. The effectiveness of peptidesand/or conjugated peptides as inhibitors of B1 activity may be evaluatedin Male green monkeys (Cercopithaecus aethiops St Kitts) challengedlocally with B1 agonists essentially as described by deBlois and Horlick(British Journal of Pharmacology. 132:327-335 (2002)), which is herebyincorporated by reference in its entirety).

In order to determine whether PEG-conjugated peptide antagonists of thepresent invention inhibit B1 induced oedema the studies described belowwere conducted on male green monkeys (Cercopithaecus aethiops St Kitts)at the Caribbean Primates Ltd. experimental farm (St Kitts, WestIndies). Procedures were reviewed and accepted by the Animal CareCommittees of the CR-CHUM (Montreal, Canada) and of Caribbean PrimatesLtd. (St Kitts, West Indies). Animals weighing 6.0±0.5 kg (n=67) wereanaesthetized (50 mg ketamine kg⁻¹) and pretreated with a singleintravenous injection of LPS (90 μg kg⁻¹) or saline (1 ml) via thesaphenous vein.

1. Inflammation Studies

Kinin-induced oedema was evaluated by the ventral skin fold assay(Sciberras et al., 1987). Briefly, anaesthetized monkeys were injectedwith captopril (1 mg kg⁻¹ 30 min before assay). A single subcutaneousinjection of dKD, BK or the vehicle (2 mM amastatin in 100 μl Ringer'slactate) was given in the ventral area and the increase in thickness ofskin folds was monitored for 30-45 min using a calibrated caliper. Theresults were expressed as the difference between the skin fold thicknessbefore and after the subcutaneous injection. Captopril and amastatinwere used to reduce degradation of kinins at the carboxyl- andamino-terminus, respectively.

Antagonist Schild Analysis

The dose-response relationship for dKD (1-100 nmol)-induced oedema wasdetermined at 24 h post-LPS in the absence or presence of differentconcentrations of PEG-peptide antagonist. BK (30 nmol) was used as apositive control.

Antagonist Time Course

The time course of inhibition by antagonist was determined at 4, 24, 48,72 and/or 96 h after single bolus administration. BK (30 nmol) was usedas a positive control.

Drugs

Ketamine hydrochloride, LPS, amastatin and captopril were from Sigma(MO, U.S.A.). All peptides were from Phoenix Pharmaceuticals (CA,U.S.A.).

Statistics

Values are presented as mean±standard error of the mean (s.e. mean). Inedema studies, the pre-injection thickness of the skin folds wassubtracted from the values after subcutaneous challenge. Curve fittingand EC₅₀ calculations were obtainedusing the Delta Graph 4.0 softwarefor Apple Computers. Data were compared by two-way analysis of variancefollowed by unpaired, one tail Student's t-test with Bonferronicorrection. p<0.05 was considered statistically significant.

LPS administration to green monkeys increased from a null level theirsensitivity to a B₁ receptor agonist in an edema formation assay.Comparatively, responses to the B₂ receptor agonist BK were notaffected.

Surprisingly, a single subcutaneous dose at 10 mg/kg of a representative5kD PEG-conjugated peptide and a 20 kD PEG-conjugated peptide of thesame peptide analog was sufficient to relieve a pre-established B1agonist induced inflammatory response and suppress successive dailyagonist challenges for 3 and 4 days, respectively. No tachyphalaxis wasobserved with B1 challenge up to 96 h. The effect was also determined tobe selective at B1 rather than B2. Furthermore, the 5K PEG-conjugateinhibited edema in response to dKD challenge longer than theunconjugated (i.e., native peptide) peptide although rapid onset andefficacy was comparable for both molecules up to 1.25 hours.

Example 9 Rat Pharmacokinetic Studies

Various peptides or conjugated peptides (in an aqueous medium) weredosed as a bolus to male Sprague-Dawley rats via an intravenous (iv) orsubcutaneous (sc) route. Blood samples were collected at various timepoints (e.g., 0, 15, 30 minutes and/or 1, 2, 4, 6, 8, 10, 12, 18, 24,30, 36, 42, 48, 60, 72, 84, 96, 120, 240, and/or 320 hours after theinjection) into heparized tubes. Plasma was removed from pelleted cellsupon centrifugation and either frozen or immediately processed. Thecompound of interest in the plasma was quantitated by ananalyte-specific LC-MS/MS or an ELISA method. Various standardpharmacokinetic parameters such as clearance (CL), apparent clearance(CL/F), volume of distribution (Vss), mean residence time (MRT), areaunder the curve (AUC), and terminal half-life (t_(1/2)) were calculatedby non-compartmental method (for example, see Table 9). TABLE 9 Peptideand PEGylated peptide B1 antagonists Pharmacokinetic Studies in RatActivated PEG Peptides t_(1/2) AUC reagent per PEG Peptide (h) 0-infMeO-20K-Maleimide^(a) 1 SEQ ID NO: 33 — 28387 MeO-20K- 1 SEQ ID NO: 1333.7^(c) 16877^(c) propionaldehyde^(b) Maleimide-20K- 2 SEQ ID NO: 3325.6^(c) 28580^(c) Maleimide^(a) MeO-20K SPA^(d) 1 SEQ ID NO: 1327.3^(c) 10701^(c) Tetrakis-20K-SPA^(d) 4 SEQ ID NO: 13 28.7^(e) 8475^(e) Tetrakis-20K-SPA^(f) 4 SEQ ID NO: 13 30.8^(e) 63239^(e) noneNA SEQ ID NO: 13  1.2^(g)  255^(g) none NA SEQ ID NO: 15  2.76^(c) 7720^(c) none NA SEQ ID NO: 13  0.6^(h)   9^(h) none NA SEQ ID NO: 49 1.0^(i) 13862^(i) none NA SEQ ID NO: 50  2.0^(i)  5529^(i) none NA SEQID NO: 22  1.0^(i)  7891^(i) None NA SEQ ID NO: 37  0.4^(i)  1122^(i)None NA SEQ ID NO: 37  0.6^(g) 18288^(g)^(a)Generated by one-pot process described in Scheme 4;^(b)Reductive amination at N-terminal residue, epsilon amine;^(c)1 mpk sc;^(d)acylated on the N-terminal residue, epsilon amine;^(e)0.5 mpk sc;^(f)acylated on the N-terminal residue, alpha amine^(g)30 mpk sc;^(h)1 mpk iv; and^(i)3 mpk iv

It will be appreciated that various modifications may be made in theinvention as described above. Accordingly, the scope of the invention isdefined in the following claims.

1. A composition of matter of the formula: F—[(X¹)—(Y¹)_(n)], or aphysiologically acceptable salt thereof, wherein: X¹ and Y¹ areindependently in each instance peptides of the formula -L¹-P¹ and-L²-P², respectively; F is a vehicle covalently bound to X¹ or Y¹; L¹and L² are independently in each instance absent or linkers having from0 to 9 amino acid residues; n is 0 to 3; and P¹ and P² are independentlyin each instance peptide antagonists of the bradykinin B1 receptor.
 2. Acomposition of matter of the formula: F′—R_(z), or a physiologicallyacceptable salt thereof, wherein: F′ is a multivalent vehicle; R isindependently in each instance —(X¹)—(Y¹ )_(n) wherein R is covalentlybound to F′; X¹ and Y¹ are independently in each instance peptides ofthe formula -L¹-P¹ and -L²-P², respectively; L¹ and L² are independentlyin each instance absent or linkers having from 0 to 9 amino acidresidues; n is 0 to 3; Z is 2 to 8; and p¹ and P² are independently ineach instance peptide antagonists of the bradykinin B1 receptor.
 3. Thecomposition of matter of any of claims 1-2 wherein P¹ and P² areindependently in each instance peptide antagonists of the bradykinin B1receptor selected from the group of peptides consisting of the peptidesshown in SEQ ID NOS: 5-26, 43-60, and derivatives thereof.
 4. Thecomposition of matter of any one of claims 1-2 wherein X¹ is a peptidehaving an amino acid sequence selected from the group consisting of thepeptides shown in SEQ ID NOS: 27-41, and derivatives thereof.
 5. Thecomposition of matter of any one of claims 1-2 wherein n is 1 and P² isa peptide having a sequence as shown in SEQ ID NO:42.
 6. The compositionof matter of any one of claims 1-2 wherein n is
 0. 7. The composition ofmatter of any one of claims 1-2 wherein P¹ and P² are independently ineach instance peptide antagonists from the group of peptides consistingof peptides defined by the formula:NH₂-a⁰a¹a²a³a⁴a⁵a⁶a⁷a⁸a⁹a¹⁰a¹¹a¹²a¹³a¹⁴-COOH wherein: a⁰ is a basic orneutral aromatic, aliphatic, heterocyclic, or alicyclic amino acid, abasic di-peptide, or absent; a¹, a², a³ and a⁴ are independently in eachinstance basic or neutral aromatic, aliphatic, heterocyclic, oralicyclic amino acids; a⁶ is Ser; a⁵, a⁷, and a⁸ are independently ineach instance aromatic, aliphatic, heterocyclic, or alicyclic aminoacids, provided that at least one of a⁵, a⁷, and a⁸ is selected fromChg, Cpg, Ig1a, Ig1b, Niga and Nigb of the D- or L-configuration; anda⁹, a¹⁰, a¹¹, a¹², a¹³, and a¹⁴ are independently in each instance anatural amino acid or absent.
 8. The composition of matter of claim 7wherein: a⁰ is a basic amino acid or basic di-peptide or absent; a¹ is abasic amino acid or basic di-peptide; a² is Pro; a³ is Hyp; a⁴ is Gly;a⁵ and a⁸ are independently in each instance Indanyl amino acids; a⁶ isSer; a⁷ is a D-Indanyl amino acid; a⁸ is Cpg; and a⁹, a¹⁰, a¹¹, a¹²,a¹³, and a¹⁴ are independently in each instance a natural amino acid orabsent.
 9. The composition of matter of claim 7 wherein: a⁰ is an aminoacid selected from the group consisting of Arg, D-Arg, Orn, D-Orn, Lys,DLys, or a di-peptide consisting of two amino acids independentlyselected from the group consisting of Arg, D-Arg, Orn, D-Orn, Lys, orDLys; a¹ is Arg; a² is Pro; a³ is Hyp; a⁴ is Gly; a⁵ is Cpg; a⁶ is Ser;a⁷ is DTic; and a⁸ is Cpg.
 10. The composition of matter of claim 7wherein: a⁰ is Lys-Lys; a¹ is Arg; a² is Pro; a³ is Hyp; a⁴ is Gly; a⁵is Ig1b; a⁶ is Ser; a⁷ is DIg1b; and a⁸ is Oic.
 11. The composition ofmatter of claim 7 wherein: a⁰ is DArg; a¹ is Arg; a² is Pro; a³ is Hyp;a⁴ is Gly; a⁵ is Igl; a⁶ is Ser; a⁷ is DIg1; and a⁸is Oic.
 12. Acomposition of matter of any one of the claims 1-2 wherein P¹ and P² areindependently in each instance peptide antagonists from the group ofpeptides consisting of peptides defined by the formula:NH₂-a⁰a¹a²a³a⁴a⁵a⁶a⁷a⁸a⁹a¹⁰a¹¹a¹²a¹³a¹⁴-COOH wherein: a¹⁰ is absent or abasic amino acid or a basic di-peptide; a¹ is Arg; a² is Pro; a³ is Pro;a⁴ is Gly; a⁵ is Me—Phe; a⁶ is Ser; a⁷ is D-β-NaI; and a⁸ is Ile. 13.The composition of matter of any one of claims 1-2 wherein said L¹ is apeptidyl linker having an amino acid sequence selected from the groupconsisting of SEQ ID NO:100 to SEQ ID NO: 105, inclusive.
 14. Thecomposition of matter of claim 3 wherein said L¹ is a peptidyl linkerhaving an amino acid sequence selected from the group consisting of SEQID NO:100 to SEQ ID NO: 105, inclusive.
 15. A method of treating,preventing, or ameliorating a disease or condition associated with ormediated by B1 activity comprising administering to a human or animalsubject a therapeutically effective amount of the composition of matteraccording to any one of claims 1-2.
 16. A method of treating,preventing, or ameliorating a disease or condition associated with ormediated by B1 activity comprising administering to a human or animalsubject a therapeutically effective amount of the composition of matteraccording to claim
 3. 17. The method according to claim 15 wherein thedisease or condition is selected from the group consisting ofinflammation, inflammatory pain, acute pain, dental pain, back pain,lower back pain, pain from trauma, surgical pain, inflammatory boweldisorders, asthma, and allergic rhinitis.
 18. The method according toclaim 16 wherein the disease or condition is selected from the groupconsisting of inflammation, inflammatory pain, acute pain, dental pain,back pain, lower back pain, pain from trauma, surgical pain,inflammatory bowel disorders, asthma, and allergic rhinitis.
 19. Apharmaceutical composition comprising a composition of matter accordingto any one of claims 1-2, and at least one pharmaceutically-acceptablediluent, excipient, or carrier.
 20. A composition of matter of theformula: F—[(X¹)—(Y¹)_(n)], or a physiologically acceptable saltthereof, wherein: X¹ and Y¹ are independently in each instance peptidesof the formula -L¹-P¹ and -L²-P², respectively; F is a PEG covalentlybound to X¹ or Y¹; L¹ and L² are independently in each instance absentor linkers having from 0 to 9 amino acid residues; n is 0 to 3; and P¹and P² are independently in each instance peptide antagonists of thebradykinin B1 receptor.
 21. The composition of matter of claim 2 whereinF′ is a multivalent PEG.
 22. The composition of matter of any one ofclaims 20-21 wherein P¹ and P² are independently in each instancepeptide antagonists of the bradykinin B1 receptor selected from thegroup of peptides consisting of the peptides shown in SEQ ID NOS: 5-60and derivatives thereof.
 23. The composition of matter of claim 21wherein X¹ is a peptide having a amino acid sequence selected from thegroup consisting of the peptides as shown in SEQ ID NOS: 27-41 andderivatives thereof.
 24. The composition of matter of claim 21 wherein nis 0 and Z is 2-8.
 25. The composition of matter of claim 24 wherein Zis
 4. 26. A composition of matter of the formula: F—[(X¹)—(Y¹)_(n)], ora physiologically acceptable salt thereof, wherein: X¹ and Y¹ areindependently in each instance peptides of the formula -L¹-P¹ and-L²-P², respectively; F is a vehicle covalently bound to X¹ or Y¹; L¹and L² are independently in each instance absent or linkers having from0 to 9 amino acid residues; n is 0 to 3; and P¹ and P² are independentlyin each instance peptide antagonists from the group of peptidesconsisting of peptides defined by the formula:NH₂-a⁰a¹a²a³a⁴a⁵a⁶a⁷a⁸a⁹a¹⁰a¹¹a¹²a¹³a¹⁴-COOH wherein: a⁰ is a basic orneutral aromatic, aliphatic, heterocyclic, or alicyclic amino acid,basic di-peptide, or absent; a¹, a², a³ and a⁴ are independently in eachinstance basic or neutral aromatic, aliphatic, heterocyclic, oralicyclic amino acids; a⁶ is Ser; a⁵, a⁷, and a⁸ are independently ineach instance aromatic, aliphatic, heterocyclic, or alicyclic aminoacids,.provided that at least one of a⁵, a⁷, and a⁸ is selected fromChg, Cpg, Ig1a, Ig1b, Niga and Nigb of the D- or L-configuration; anda⁹, a¹⁰, a¹¹, a¹², a¹³, and a¹⁴ are independently in each instance anatural amino acid or absent.
 27. A composition of matter of theformula: F′—R_(z), or a physiologically acceptable salt thereof,wherein: F′ is a multivalent vehicle; R is independently in eachinstance —(X¹)—(Y¹)_(n) wherein R is covalently bound to F′; X¹ and Y¹are independently in each instance peptides of the formula -L′-P¹ and-L²-P², respectively; L¹ and L² are independently in each instanceabsent or linkers having from 0 to 9 amino acid residues; n is 0 to 3; Zis 2 to 8; and P¹ and P² are independently in each instance peptideantagonists from the group of peptides consisting of peptides defined bythe formula:NH₂-a⁰a¹a²a³a⁴a⁵a⁶a⁷a⁸a⁹a¹⁰a¹¹a¹²a¹³a¹⁴-COOH wherein: a⁰ is a basic orneutral aromatic, aliphatic, heterocyclic, or alicyclic amino acid,basic di-peptide, or absent; a¹, a², a^(3,) and a⁴ are independently ineach instance basic or neutral aromatic, aliphatic, heterocyclic, oralicyclic amino acids; a⁶ is Ser; a⁵, a⁷, and a⁸ are independently ineach instance aromatic, aliphatic, heterocyclic, or alicyclic aminoacids, provided that at least one of a⁵, a⁷, and a⁸ is selected fromChg, Cpg, Ig1a, Ig1b, Niga and Nigb of the D- or L-configuration; anda⁹, a¹⁰, a¹¹, a¹², a¹³, and a¹⁴ are independently in each instance anatural amino acid or absent.
 28. The composition of matter of any oneof claims 26-27 wherein: a⁰ is a basic amino acid, a basic di-peptide,or absent; a¹ is a basic amino acid; a² is Pro; a³ is Hyp; a⁴ is Gly; a⁵and a⁸ are independently in each instance Indanyl amino acids; a⁶ isSer; a⁷ is a D-Indanyl amino acid; a⁸ is Cpg; and a⁹, a¹⁰, a¹¹, a¹²,a¹³, and a¹⁴ are independently in each instance a amino acid or absent.29. The composition of matter of any one of claims 26-27 wherein: a⁰ isan amino acid selected from the group consisting of Arg, D-Arg, Orn,D-Orn, Lys, DLys, or a di-peptide consisting of two amino acidsindependently selected from the group consisting of Arg, D-Arg, Orn,D-Orn, Lys, or DLys; a¹ is Arg; a² is Pro; a³ is Hyp; a⁴ is Gly; a⁵ isCpg; a⁶ is Ser; a⁷ is DTic; and a⁸ is Cpg.
 30. The composition of matterof any one of claims 26-27 wherein: a⁰ is D-Arg, or a dipeptide selectedfrom the group consisting of Lys-Lys, DLys-Lys, and DOrn-Lys; a¹ is Arg;a² is Pro; a³ is Hyp; a⁴ is Gly; a⁵ is Ig1b; a⁶ is Ser; a⁷ is DIg1b; anda⁸ is Oic.
 31. The composition of matter of any one of claims 26-27wherein: a⁰ is DArg or a Lys-Lys di-peptide; a¹ is Arg; a² is Pro; a³ isHyp; a⁴ is Gly; a⁵ is Ig1; a⁶ is Ser; a⁷ is DIg1; and a⁸ is Oic.
 32. Acomposition of matter of any one of claims 1-2 wherein P¹ and P² areindependently in each instance peptide antagonists from the group ofpeptides consisting of peptides defined by the formula:NH₂-a⁰a¹a²a³a⁴a⁵a⁶a⁷a⁸a⁹a¹⁰a¹¹a¹²a¹³a¹⁴-COOH wherein: a⁰ is an aminoacid selected from the group consisting of Arg, D-Arg, Orn, D-Orn, Lys,DLys, or a dipeptide consisting of two amino acids independentlyselected from the group consisting of Arg, D-Arg, Orn, D-Orn, Lys, orDLys; a¹ is Arg; a² is Pro; a³ is Pro; a⁴ is Gly; a⁵ is Me—Phe; a⁶ isSer; a⁷ is D-β-NaI; and a⁸ is Ile.
 33. The composition of matter ofclaim 32 wherein said L¹ is a peptidyl linker having an amino acidsequence selected from the group consisting of SEQ ID NO:100 to SEQ IDNO: 105, inclusive.
 34. The composition of matter of claim 33 whereinsaid polyethylene glycol has an average molecular weight selected fromthe group consisting of: i. 5,000 Daltons; ii. 20,000 Daltons; iii.24,000 Daltons; iv. 30,000 Daltons; v. 40,000 Daltons; and vi. 60,000Daltons.
 35. The composition of matter of claim 34 wherein saidcomposition of matter is capable of antagonizing B1 receptor activity invitro and has a therapeutically acceptable half-life in vivo in mammals.36. A method of treating, preventing, or ameliorating a disease orcondition associated with or mediated by B1 activity comprisingadministering to a human or animal subject a therapeutically effectiveamount of the composition of matter according to claim
 34. 37. Themethod according to claim 35 wherein the disease or condition isselected from the group consisting of inflammation, inflammatory pain,acute pain, dental pain, back pain, lower back pain, pain from trauma,surgical pain, inflammatory bowel disorders, asthma, and allergicrhinitis.
 38. A pharmaceutical composition comprising the composition ofmatter according to claim 34 and at least onepharmaceutically-acceptable diluent, excipient, or carrier.
 39. The useof the composition of matter according to claim 34 in the manufacture ofa medicament for the treatment of a disease or condition selected fromthe group consisting of inflammation, inflammatory pain, acute pain,dental pain, back pain, lower back pain, pain from trauma, surgicalpain, inflammatory bowel disorders, asthma, and allergic rhinitis.
 40. Apeptide antagonist of the bradykinin B1 receptor comprising a sequenceof amino acids selected from the group consisting of SEQ ID NOS: 15-35,39-54, derivatives thereof, and a physiologically acceptable saltthereof.
 41. A peptide antagonist of the bradykinin B1 receptorcomprising a sequence of amino acids selected from the group consistingof SEQ ID NOS: 15-35, or a physiologically acceptable salt thereof. 42.The peptide of claim 41 comprising a sequence of amino acids selectedfrom the group consisting of SEQ ID NOS: 15-26.
 43. The peptide of claim40 wherein the N-terminal amino acid is a D-amino acid.
 44. The peptideof claim 43 wherein the N-terminal amino acid is selected from the groupconsisting of D-Dab, DLys, DArg, and DOrn.
 45. A pharmaceuticalcomposition comprising a peptide antagonist of the bradykinin B1receptor comprising a sequence of amino acids selected from the groupconsisting of SEQ ID NOS: 15-35 and at least onepharmaceutically-acceptable diluent, excipient, or carrier.
 46. A methodof treating, preventing, or ameliorating a disease or conditionassociated with or mediated by B1 activity comprising administering to ahuman or animal subject a therapeutically effective amount of apharmaceutical composition comprising a peptide antagonist of thebradykinin B1 receptor comprising a sequence of amino acids selectedfrom the group consisting of SEQ ID NOS: 15-35 and at least onepharmaceutically-acceptable diluent, excipient, or carrier.
 47. Themethod according to claim 46 wherein the disease or condition isselected from the group consisting of inflammation, inflammatory pain,acute pain, dental pain, back pain, lower back pain, pain from trauma,surgical pain, inflammatory bowel disorders, asthma, and allergicrhinitis.